Production of galactosylated di- and oligosaccharides

ABSTRACT

The disclosure is in the technical field of synthetic biology and metabolic engineering. Described is the use of a new type of galactosyltransferases for the production of a galactosylated di- or oligosaccharide. The disclosure also describes methods for the production of a galactosylated di- or oligosaccharide as well as the purification of the di- or oligosaccharide. Furthermore, the disclosure is in the field of cultivation or fermentation of metabolically engineered cells. The disclosure provides a cell metabolically engineered for production of a galactosylated di- or oligosaccharide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International PatentApplication PCT/EP2021/072270, filed Aug. 10, 2021, designating theUnited States of America and published as International PatentPublication WO 2022/034076 A2 on Feb. 17, 2022, which claimed thebenefit under Article 8 of the Patent Cooperation Treaty to EuropeanPatent Application Serial Nos. 21186202.4 and 21186203.2 filed Jul. 16,2021; to European Patent Application Serial No. 21168997.1 filed Apr.16, 2021; and to European Patent Application Serial Nos. 20190198.0,20190200.4, 20190201.2, 20190202.0, 20190203.8, 20190204.6, 20190205.3,20190206.1, 20190207.9, and 20190208.7, all filed on Aug. 10, 2020, thedisclosure of each of which is hereby incorporated herein in itsentirety by this reference.

TECHNICAL FIELD

The application is in the technical field of synthetic biology andmetabolic engineering. Described is the use of a new type ofgalactosyltransferases for the production of a galactosylated di- oroligosaccharide. Also described are methods for producing agalactosylated di- or oligosaccharide as well as the purification of thedi- or oligosaccharide. Furthermore, the disclosure is in the field ofcultivation or fermentation of metabolically engineered cells. Thedisclosure provides a cell metabolically engineered for production of agalactosylated di- or oligosaccharide.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

Pursuant to 37 C.F.R. § 1.821, a Sequence Listing ASCII text fileentitled “4006-P17268US_ST26.xml,” 102,741 bytes in size, generated Feb.10, 2023, has been submitted via EFS-Web is provided in lieu of a papercopy. This Sequence Listing is hereby incorporated by reference into thespecification for its disclosures.

BACKGROUND

Disaccharides and oligosaccharides, often present as glyco-conjugatedforms to proteins and lipids, are involved in many vital phenomena suchas differentiation, development and biological recognition processesrelated to the development and progress of fertilization, embryogenesis,inflammation, metastasis and host pathogen adhesion. Oligosaccharidescan also be present as unconjugated glycans in body fluids and humanmilk wherein they also modulate important developmental andimmunological processes (Bode, Early Hum. Dev. 1-4 (2015); Reily et al.,Nat. Rev. Nephrol. 15, 346-366 (2019); Varki, Glycobiology 27, 3-49(2017)). There is large scientific and commercial interest ingalactosylated di- and oligosaccharides due to the wide functionalspectrum of these saccharides. Yet, the availability of galactosylateddi- and oligosaccharides is limited as production relies on chemical orchemo-enzymatic synthesis or on purification from natural sources suchas, e.g., animal milk. Chemical synthesis methods are laborious andtime-consuming and because of the large number of steps involved theyare difficult to scale-up. Enzymatic approaches usingglycosyltransferases offer many advantages above chemical synthesis.Glycosyltransferases catalyze the transfer of a sugar moiety from anactivated nucleotide-sugar donor onto saccharide or non-saccharideacceptors (Coutinho et al., J. Mol. Biol. 328 (2003) 307-317). Theseglycosyltransferases are the source for biotechnologists to synthesizeoligosaccharides and are used both in (chemo)enzymatic approaches aswell as in cell-based production systems. However, stereospecificity andregioselectivity of glycosyltransferases and of galactosyltransferases,as part of the glycosyltransferase family, are still a formidablechallenge. In addition, chemo-enzymatic approaches need to regenerate insitu nucleotide-sugar donors. Cellular production of di- andoligosaccharides needs tight control of spatiotemporal availability ofadequate levels of nucleotide-sugar donors in proximity of complementaryglycosyltransferases.

BRIEF SUMMARY

Provided are tools and methods by means of which a galactosylated di- oroligosaccharide can be produced in an efficient, time and cost-effectiveway and if needed, continuous process.

Provided is the use of a new type of galactosyltransferase for theproduction of a galactosylated di- or oligosaccharide, methods and acell for the production of a galactosylated di- or oligosaccharide,wherein the cell is genetically modified for the production of thegalactosylated di- or oligosaccharide.

Surprisingly, it has now been found that it is possible to produce agalactosylated di- or oligosaccharide with a new type ofgalactosyltransferases, more specifically a new type ofN-acetylglucosamine b-1,X-galactosyltransferases. The disclosureprovides use of a new type of N-acetylglucosamineb-1,3-galactosyltransferases and N-acetylglucosamineb-1,4-galactosyltransferases that galactosylate acceptors like anN-acetylglucosamine and/or N-acetylgalactosamine as a monosaccharideand/or that galactosylate acceptors like an N-acetylglucosamine and/orN-acetylgalactosamine as part of a di- and/or oligosaccharide at thenon-reducing end of the oligosaccharide. The disclosure provides the useof the N-acetylglucosamine b-1,X-galactosyltransferases to produce agalactosylated di- or oligosaccharide. The disclosure provides methodsand a cell for the production of a galactosylated di- or oligosaccharideby using the galactosyltransferases. The methods comprise providingUDP-galactose and any one of the new galactosyltransferases, andcontacting any one of the galactosyltransferases and UDP-galactose withone or more acceptor(s), under conditions where thegalactosyltransferase catalyzes the galactosylation of the acceptor(s).One method comprises the steps of providing a cell that is capable ofsynthesizing UDP-galactose and any one or more of the acceptor(s) andthat is capable of expressing any one of the galactosyltransferasescapable of galactosylating the acceptors, and cultivation of the cellunder conditions permissive for producing the galactosylated di- oroligosaccharide. Next, the disclosure also provides methods to separatethe galactosylated di- or oligosaccharide. Furthermore, the disclosureprovides a cell metabolically engineered for production of agalactosylated di- or oligosaccharide.

Definitions

The words used in this specification to describe the disclosure and itsvarious embodiments are to be understood not only in the sense of theircommonly defined meanings, but to include by special definition in thisspecification structure, material or acts beyond the scope of thecommonly defined meanings. Thus, if an element can be understood in thecontext of this specification as including more than one meaning, thenits use in a claim must be understood as being generic to all possiblemeanings supported by the specification and by the word itself.

The various embodiments and aspects of the disclosure disclosed hereinare to be understood not only in the order and context specificallydescribed in this specification, but to include any order and anycombination thereof. Whenever the context requires, all words used inthe singular number shall be deemed to include the plural and viceversa. Unless defined otherwise, all technical and scientific terms usedherein generally have the same meaning as commonly understood by one ofordinary skill in the art to which this disclosure belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization described herein are those well-known andcommonly employed in the art. Standard techniques are used for nucleicacid and peptide synthesis. Generally, purification steps are performedaccording to the manufacturer's specifications.

In the specification, there have been disclosed embodiments of thedisclosure, and although specific terms are employed, the terms are usedin a descriptive sense only and not for purposes of limitation, thescope of the disclosure being set forth in the following claims. It mustbe understood that the illustrated embodiments have been set forth onlyfor the purposes of example and that it should not be taken as limitingthe disclosure. It will be apparent to those skilled in the art thatalterations, other embodiments, improvements, details and uses can bemade consistent with the letter and spirit of the disclosure herein andwithin the scope of this disclosure, which is limited only by theclaims, construed in accordance with the patent law, including thedoctrine of equivalents. In the claims that follow, reference charactersused to designate claim steps are provided for convenience ofdescription only, and are not intended to imply any particular order forperforming the steps.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. Throughout the application, the verb “to comprise” maybe replaced by “to consist” or “to consist essentially of” and viceversa. In addition the verb “to consist” may be replaced by “to consistessentially of” meaning that a composition as defined herein maycomprise additional component(s) than the ones specifically identified,the additional component(s) not altering the unique characteristic ofthe disclosure. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one.”

Throughout the application, unless explicitly stated otherwise, thearticles “a” and “an” are preferably replaced by “at least two,” morepreferably by “at least three,” even more preferably by “at least four,”even more preferably by “at least five,” even more preferably by “atleast six,” most preferably by “at least two.”

Each embodiment as identified herein may be combined together unlessotherwise indicated. All publications, patents, and patent applicationsmentioned in this specification are herein incorporated by reference tothe same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference. The full content of the priorityapplications, including EP20190198, EP20190200 and EP20190207, is alsoincorporated by reference to the same extent as if the priorityapplication was specifically and individually indicated to beincorporated by reference.

According to the disclosure, the term “polynucleotide(s)” generallyrefers to any polyribonucleotide or polydeoxyribonucleotide, which maybe unmodified RNA or DNA or modified RNA or DNA. “Polynucleotide(s)”include, without limitation, single- and double-stranded DNA, DNA thatis a mixture of single- and double-stranded regions or single-, double-and triple-stranded regions, single- and double-stranded RNA, and RNAthat is mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded, or triple-stranded regions, or a mixture of single- anddouble-stranded regions. In addition, “polynucleotide” as used hereinrefers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The strands in such regions may be from the same molecule or fromdifferent molecules. The regions may include all of one or more of themolecules, but more typically involve only a region of some of themolecules. One of the molecules of a triple-helical region often is anoligonucleotide. As used herein, the term “polynucleotide(s)” alsoincludes DNAs or RNAs as described above that contain one or moremodified bases. Thus, DNAs or RNAs with backbones modified for stabilityor for other reasons are “polynucleotide(s)” according to thedisclosure. Moreover, DNAs or RNAs comprising unusual bases, such asinosine, or modified bases, such as tritylated bases, are to beunderstood to be covered by the term “polynucleotides.” It will beappreciated that a great variety of modifications have been made to DNAand RNA that serve many useful purposes known to those of skill in theart. The term “polynucleotide(s)” as it is employed herein embraces suchchemically, enzymatically or metabolically modified forms ofpolynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including, for example, simple andcomplex cells. The term “polynucleotide(s)” also embraces shortpolynucleotides often referred to as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to the skilled person. The same typeof modification may be present in the same or varying degree at severalsites in a given polypeptide. Furthermore, a given polypeptide maycontain many types of modifications. Modifications can occur anywhere ina polypeptide, including the peptide backbone, the amino acidsidechains, and the amino or carboxyl termini. Modifications include,for example, acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation, selenoylation, transfer-RNA mediated addition of aminoacids to proteins, such as arginylation, and ubiquitination.Polypeptides may be branched or cyclic, with or without branching.Cyclic, branched and branched circular polypeptides may result frompost-translational natural processes and may be made by entirelysynthetic methods, as well.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the disclosure. The term also encompasses polynucleotidesthat include a single continuous region or discontinuous regionsencoding the polypeptide (for example, interrupted by integrated phageor an insertion sequence or editing) together with additional regionsthat also may contain coding and/or non-coding sequences.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated,” as the term is employedherein. Similarly, a “synthetic” sequence, as the term is used herein,means any sequence that has been generated synthetically and notdirectly isolated from a natural source. “Synthesized,” as the term isused herein, means any synthetically generated sequence and not directlyisolated from a natural source.

The terms “recombinant” or “transgenic” or “metabolically engineered” or“genetically modified,” as used herein with reference to a cell or hostcell are used interchangeably and indicates that the cell replicates aheterologous nucleic acid, or expresses a peptide or protein encoded bya heterologous nucleic acid (i.e., a sequence “foreign to the cell” or asequence “foreign to the location or environment in the cell”). Suchcells are described to be transformed with at least one heterologous orexogenous gene, or are described to be transformed by the introductionof at least one heterologous or exogenous gene. Metabolically engineeredor recombinant or transgenic cells can contain genes that are not foundwithin the native (non-recombinant) form of the cell. Recombinant cellscan also contain genes found in the native form of the cell wherein thegenes are modified and re-introduced into the cell by artificial means.The terms also encompass cells that contain a nucleic acid endogenous tothe cell that has been modified or its expression or activity has beenmodified without removing the nucleic acid from the cell; suchmodifications include those obtained by gene replacement, replacement ofa promoter; site-specific mutation; and related techniques. Accordingly,a “recombinant polypeptide” is one that has been produced by arecombinant cell. A “heterologous sequence” or a “heterologous nucleicacid,” as used herein, is one that originates from a source foreign tothe particular cell (e.g., from a different species), or, if from thesame source, is modified from its original form or place in the genome.Thus, a heterologous nucleic acid operably linked to a promoter is froma source different from that from which the promoter was derived, or, iffrom the same source, is modified from its original form or place in thegenome. The heterologous sequence may be stably introduced, e.g., bytransfection, transformation, conjugation or transduction, into thegenome of the host microorganism cell, wherein techniques may be appliedthat will depend on the cell and the sequence that is to be introduced.Various techniques are known to a person skilled in the art and are,e.g., disclosed in Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989). The term “mutant” cell or microorganism as usedwithin the context of the disclosure refers to a cell or microorganismthat is genetically modified.

The terms “cell genetically modified for the production of agalactosylated di- or oligosaccharide” within the context of thedisclosure refers to a cell of a microorganism that is geneticallymodified in the expression or activity of one or more enzyme(s) selectedfrom the group comprising: glucosamine 6-phosphate N-acetyltransferase,phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphateaminotransferase, and UDP-glucose 4-epimerase.

The term “endogenous,” within the context of the disclosure refers toany polynucleotide, polypeptide or protein sequence that is a naturalpart of a cell and is occurring at its natural location in the cellchromosome and of which the control of expression has not been alteredcompared to the natural control mechanism acting on its expression. Theterm “exogenous” refers to any polynucleotide, polypeptide or proteinsequence that originates from outside the cell under study and not anatural part of the cell or that is not occurring at its naturallocation in the cell chromosome or plasmid.

The term “heterologous” when used in reference to a polynucleotide,gene, nucleic acid, polypeptide, or enzyme refers to a polynucleotide,gene, nucleic acid, polypeptide, or enzyme that is from a source orderived from a source other than the host organism species. In contrasta “homologous” polynucleotide, gene, nucleic acid, polypeptide, orenzyme is used herein to denote a polynucleotide, gene, nucleic acid,polypeptide, or enzyme that is derived from the host organism species.When referring to a gene regulatory sequence or to an auxiliary nucleicacid sequence used for maintaining or manipulating a gene sequence(e.g., a promoter, a 5′ untranslated region, 3′ untranslated region,poly A addition sequence, intron sequence, splice site, ribosome bindingsite, internal ribosome entry sequence, genome homology region,recombination site, etc.), “heterologous” means that the regulatorysequence or auxiliary sequence is not naturally associated with the genewith which the regulatory or auxiliary nucleic acid sequence isjuxtaposed in a construct, genome, chromosome, or episome. Thus, apromoter operably linked to a gene to which it is not operably linked toin its natural state (i.e., in the genome of a non-geneticallyengineered organism) is referred to herein as a “heterologous promoter,”even though the promoter may be derived from the same species (or, insome cases, the same organism) as the gene to which it is linked.

The term “modified activity” of a protein or an enzyme relates to achange in activity of the protein or the enzyme compared to the wildtype, i.e., natural, activity of the protein or enzyme. The modifiedactivity can either be an abolished, impaired, reduced or delayedactivity of the protein or enzyme compared to the wild type activity ofthe protein or the enzyme but can also be an accelerated or an enhancedactivity of the protein or the enzyme compared to the wild type activityof the protein or the enzyme. A modified activity of a protein or anenzyme is obtained by modified expression of the protein or enzyme or isobtained by expression of a modified, i.e., mutant form of the proteinor enzyme. A modified activity of an enzyme further relates to amodification in the apparent Michaelis constant Km and/or the apparentmaximal velocity (Vmax) of the enzyme.

The term “modified expression” of a gene relates to a change inexpression compared to the wild type expression of the gene in any phaseof the production process of the encoded protein. The modifiedexpression is either a lower or higher expression compared to the wildtype, wherein the term “higher expression” is also defined as“overexpression” of the gene in the case of an endogenous gene or“expression” in the case of a heterologous gene that is not present inthe wild type strain. Lower expression or reduced expression is obtainedby means of common well-known technologies for a skilled person (e.g.,the usage of siRNA, CrispR, CrispRi, riboswitches, recombineering,homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA,mutating genes, knocking-out genes, transposon mutagenesis, etc.) thatare used to change the genes in such a way that they are less-able(i.e., statistically significantly “less-able” compared to a functionalwild-type gene) or completely unable (e.g., knocked-out genes) toproduce functional final products. The term “riboswitch” as used hereinis defined to be part of the messenger RNA that folds into intricatestructures that block expression by interfering with translation.Binding of an effector molecule induces conformational change(s)permitting regulated expression post-transcriptionally. Next to changingthe gene of interest in such a way that lower expression is obtained asdescribed above, lower expression can also be obtained by changing thetranscription unit, the promoter, an untranslated region, the ribosomebinding site, the Shine Dalgarno sequence or the transcriptionterminator. Lower expression or reduced expression can, for instance, beobtained by mutating one or more base pairs in the promoter sequence orchanging the promoter sequence fully to a constitutive promoter with alower expression strength compared to the wild type or an induciblepromoter that result in regulated expression or a repressible promoterthat results in regulated expression. Overexpression or expression isobtained by means of common well-known technologies for a skilled person(e.g., the usage of artificial transcription factors, de novo design ofa promoter sequence, ribosome engineering, introduction orre-introduction of an expression module at euchromatin, usage ofhigh-copy-number plasmids), wherein the gene is part of a“transcriptional unit” that relates to any sequence in which a promotersequence, untranslated region sequence (containing either a ribosomebinding sequence, Shine Dalgarno or Kozak sequence), a coding sequenceand optionally a transcription terminator is present, and leading to theexpression of a functional active protein. The expression is eitherconstitutive or regulated.

The term “constitutive expression” is defined as expression that is notregulated by transcription factors other than the subunits of RNApolymerase (e.g., the bacterial sigma factors) under certain growthconditions. Non-limiting examples of such transcription factors are CRP,LacI, ArcA, Cra, IclR in E. coli. These transcription factors bind on aspecific sequence and may block or enhance expression in certain growthconditions. RNA polymerase binds a specific sequence to initiatetranscription, for instance, via a sigma factor in prokaryotic hosts.

The term “regulated expression” is defined as expression that isregulated by transcription factors other than the subunits of RNApolymerase (e.g., bacterial sigma factors) under certain growthconditions. Examples of such transcription factors are described above.Commonly expression regulation is obtained by means of an inducer orrepressor, such as but not limited to IPTG, arabinose, rhamnose, fucose,allo-lactose or pH shifts, or temperature shifts or carbon depletion orsubstrates or the produced product or chemical repression.

The term “expression by a natural inducer” is defined as a facultativeor regulatory expression of a gene that is only expressed upon a certainnatural condition of the host (e.g., organism being in labor, or duringlactation), as a response to an environmental change (e.g., includingbut not limited to, hormone, heat, cold, pH shifts, light, oxidative orosmotic stress/signaling), or dependent on the position of thedevelopmental stage or the cell cycle of the host cell including, butnot limited to, apoptosis and autophagy.

The term “inducible expression upon chemical treatment” is defined as afacultative or regulatory expression of a gene that is only expressedupon treatment with a chemical inducer or repressor, wherein the inducerand repressor comprise but are not limited to an alcohol (e.g., ethanol,methanol), a carbohydrate (e.g., glucose, galactose, glycerol, lactose,arabinose, rhamnose, fucose, allo-lactose), metal ions (e.g., aluminum,copper, zinc), nitrogen, phosphates, IPTG, acetate, formate, xylene.

The term “control sequences” refers to sequences recognized by the cellstranscriptional and translational systems, allowing transcription andtranslation of a polynucleotide sequence to a polypeptide. Such DNAsequences are thus necessary for the expression of an operably linkedcoding sequence in a particular cell or organism. Such control sequencescan be, but are not limited to, promoter sequences, ribosome bindingsequences, Shine Dalgarno sequences, Kozak sequences, transcriptionterminator sequences. The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, and a ribosome binding site. Eukaryotic cells are known toutilize promoters, polyadenylation signals, and enhancers. DNA for apresequence or secretory leader may be operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. The control sequences can furthermore becontrolled with external chemicals, such as, but not limited to, IPTG,arabinose, lactose, allo-lactose, rhamnose or fucose via an induciblepromoter or via a genetic circuit that either induces or represses thetranscription or translation of the polynucleotide to a polypeptide.

Generally, “operably linked” means that the DNA sequences being linkedare contiguous, and, in the case of a secretory leader, contiguous andin reading phase. However, enhancers do not have to be contiguous.

The term “wild type” refers to the commonly known genetic orphenotypical situation as it occurs in nature.

The term “modified expression of a protein” as used herein refers to i)higher expression or overexpression of an endogenous protein, ii)expression of a heterologous protein or iii) expression and/oroverexpression of a variant protein that has a higher activity comparedto the wild-type (i.e., native) protein.

As used herein, the term “mammary cell(s)” generally refers to mammaryepithelial cell(s), mammary-epithelial luminal cell(s), or mammalianepithelial alveolar cell(s), or any combination thereof. As used herein,the term “mammary-like cell(s)” generally refers to cell(s) having aphenotype/genotype similar (or substantially similar) to natural mammarycell(s) but is/are derived from non-mammary cell source(s). Suchmammary-like cell(s) may be engineered to remove at least one undesiredgenetic component and/or to include at least one predetermined geneticconstruct that is typical of a mammary cell. Non-limiting examples ofmammary-like cell(s) may include mammary epithelial-like cell(s),mammary epithelial luminal-like cell(s), non-mammary cell(s) thatexhibits one or more characteristics of a cell of a mammary celllineage, or any combination thereof. Further non-limiting examples ofmammary-like cell(s) may include cell(s) having a phenotype similar (orsubstantially similar) to natural mammary cell(s), or more particularlya phenotype similar (or substantially similar) to natural mammaryepithelial cell(s). A cell with a phenotype or that exhibits at leastone characteristic similar to (or substantially similar to) a naturalmammary cell or a mammary epithelial cell may comprise a cell (e.g.,derived from a mammary cell lineage or a non-mammary cell lineage) thatexhibits either naturally, or has been engineered to, be capable ofexpressing at least one milk component.

As used herein, the term “non-mammary cell(s)” may generally include anycell of non-mammary lineage. In the context of the disclosure, anon-mammary cell can be any mammalian cell capable of being engineeredto express at least one milk component. Non-limiting examples of suchnon-mammary cell(s) include hepatocyte(s), blood cell(s), kidneycell(s), cord blood cell(s), epithelial cell(s), epidermal cell(s),myocyte(s), fibroblast(s), mesenchymal cell(s), or any combinationthereof. In some instances, molecular biology and genome editingtechniques can be engineered to eliminate, silence, or attenuate myriadgenes simultaneously.

Throughout the application, unless explicitly stated otherwise, theexpressions “capable of . . . <verb>” and “capable to . . . <verb>” maybe replaced with the active voice of the verb and vice versa. Forexample, the expression “capable of expressing” is preferably replacedwith “expresses” and vice versa, i.e., “expresses” is preferablyreplaced with “capable of expressing.”

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to thepersons skilled in the art.

The term “derivative” of a polypeptide, as used herein, is a polypeptidethat may contain deletions, additions or substitutions of amino acidresidues within the amino acid sequence of the polypeptide, but thatresult in a silent change, thus producing a functionally equivalentpolypeptide. Amino acid substitutions may be made based on similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; planar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid. Within the contextof this disclosure, a derivative polypeptide as used herein, refers to apolypeptide capable of exhibiting a substantially similar in vivoactivity as the original polypeptide as judged by any of a number ofcriteria, including but not limited to enzymatic activity, and that maybe differentially modified during or after translation. Furthermore,non-classical amino acids or chemical amino acid analogues can beintroduced as a substitution or addition into the original polypeptidesequence.

In some embodiments, the disclosure contemplates making functionalvariants by modifying the structure of an enzyme as used in thedisclosure. Variants can be produced by amino acid substitution,deletion, addition, or combinations thereof. For instance, it isreasonable to expect that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid (e.g., conservative mutations) will not have a majoreffect on the biological activity of the resulting molecule.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Whether a change inthe amino acid sequence of a polypeptide of the disclosure results in afunctional homolog can be readily determined by assessing the ability ofthe variant polypeptide to produce a response in cells in a fashionsimilar to the wild-type polypeptide.

The term “functional homolog” as used herein describes those moleculesthat have sequence similarity (in other words, homology) and also shareat least one functional characteristic such as a biochemical activity(Altenhoff et al., PLoS Comput. Biol. 8 (2012) e1002514). Functionalhomologs will typically give rise to the same characteristics to asimilar, but not necessarily the same, degree. Functionally homologousproteins give the same characteristics where the quantitativemeasurement produced by one homolog is at least 10 percent of the other;more typically, at least 20 percent, between about 30 percent and about40 percent; for example, between about 50 percent and about 60 percent;between about 70 percent and about 80 percent; or between about 90percent and about 95 percent; between about 98 percent and about 100percent, or greater than 100 percent of that produced by the originalmolecule. Thus, where the molecule has enzymatic activity the functionalhomolog will have the above-recited percent enzymatic activitiescompared to the original enzyme. Where the molecule is a DNA-bindingmolecule (e.g., a polypeptide) the homolog will have the above-recitedpercentage of binding affinity as measured by weight of bound moleculecompared to the original molecule.

A functional homolog and the reference polypeptide may be naturallyoccurring polypeptides, and the sequence similarity may be due toconvergent or divergent evolutionary events. Functional homologs aresometimes referred to as orthologs, where “ortholog,” refers to ahomologous gene or protein that is the functional equivalent of thereferenced gene or protein in another species. Orthologous genes arehomologous genes in different species that originate by vertical descentfrom a single gene of the last common ancestor, wherein the gene and itsmain function are conserved. A homologous gene is a gene inherited intwo species by a common ancestor.

The term “ortholog” when used in reference to an amino acid ornucleotide/nucleic acid sequence from a given species refers to the sameamino acid or nucleotide/nucleic acid sequence from a different species.It should be understood that two sequences are orthologs of each otherwhen they are derived from a common ancestor sequence via linear descentand/or are otherwise closely related in terms of both their sequence andtheir biological function. Orthologs will usually have a high degree ofsequence identity but may not (and often will not) share 100% sequenceidentity.

Paralogous genes are homologous genes that originate by a geneduplication event. Paralogous genes often belong to the same species,but this is not necessary. Paralogs can be split into in-paralogs(paralogous pairs that arose after a speciation event) and out-paralogs(paralogous pairs that arose before a speciation event). Between speciesout-paralogs are pairs of paralogs that exist between two organisms dueto duplication before speciation. Within species out-paralogs are pairsof paralogs that exist in the same organism, but whose duplication eventhappened after speciation. Paralogs typically have the same or similarfunction.

Functional homologs can be identified by analysis of nucleotide andpolypeptide sequence alignments. For example, performing a query on adatabase of nucleotide or polypeptide sequences can identify homologs ofthe polypeptide of interest like, e.g., a biomass-modulatingpolypeptide, a glycosyltransferase, a protein involved innucleotide-activated sugar synthesis or a membrane transporter protein.Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLASTanalysis of non-redundant databases using amino acid sequence of abiomass-modulating polypeptide, a glycosyltransferase, a proteininvolved in nucleotide-activated sugar synthesis or a membranetransporter protein, respectively, as the reference sequence. Amino acidsequence is, in some instances, deduced from the nucleotide sequence.Typically, those polypeptides in the database that have greater than 40percent sequence identity are candidates for further evaluation forsuitability as a biomass-modulating polypeptide, a glycosyltransferase,a protein involved in nucleotide-activated sugar synthesis or a membranetransporter protein, respectively. Amino acid sequence similarity allowsfor conservative amino acid substitutions, such as substitution of onehydrophobic residue for another or substitution of one polar residue foranother or substitution of one acidic amino acid for another orsubstitution of one basic amino acid for another etc. Preferably, byconservative substitutions is intended combinations such as glycine byalanine and vice versa; valine, isoleucine and leucine by methionine andvice versa; aspartate by glutamate and vice versa; asparagine byglutamine and vice versa; serine by threonine and vice versa; lysine byarginine and vice versa; cysteine by methionine and vice versa; andphenylalanine and tyrosine by tryptophan and vice versa. If desired,manual inspection of such candidates can be carried out in order tonarrow the number of candidates to be further evaluated. Manualinspection can be performed by selecting those candidates that appear tohave domains present in productivity-modulating polypeptides, e.g.,conserved functional domains.

“Fragment,” with respect to a polynucleotide, refers to a clone or anypart of a polynucleotide molecule, particularly a part of apolynucleotide that retains a usable, functional characteristic of thefull-length polynucleotide molecule. Useful fragments includeoligonucleotides and polynucleotides that may be used in hybridizationor amplification technologies or in the regulation of replication,transcription or translation. A “polynucleotide fragment” refers to anysubsequence of a polynucleotide SEQ ID NO (or GenBank NO.), typically,comprising or consisting of at least about 9, 10, 11, 12 consecutivenucleotides, for example, at least about 30 nucleotides or at leastabout 50 nucleotides of any of the polynucleotide sequences providedherein. Exemplary fragments can additionally or alternatively includefragments that comprise, consist essentially of, or consist of a regionthat encodes a conserved family domain of a polypeptide. Exemplaryfragments can additionally or alternatively include fragments thatcomprise a conserved domain of a polypeptide. As such, a fragment of apolynucleotide SEQ ID NO (or GenBank NO.) preferably means a nucleotidesequence that comprises or consists of the polynucleotide SEQ ID NO (orGenBank NO.) wherein no more than 200, 150, 100, 50 or 25 consecutivenucleotides are missing, preferably no more than 50 consecutivenucleotides are missing, and that retains a usable, functionalcharacteristic (e.g., activity) of the full-length polynucleotidemolecule that can be assessed by the skilled person through routineexperimentation. Alternatively, a fragment of a polynucleotide SEQ ID NO(or GenBank NO.) preferably means a nucleotide sequence that comprisesor consists of an amount of consecutive nucleotides from thepolynucleotide SEQ ID NO (or GenBank NO.) and wherein the amount ofconsecutive nucleotides is at least 50.0%, 60.0%, 70.0%, 80.0%, 81.0%,82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%,92.0%, 93.0%, 94.0%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%,98.5%, 99.0%, 99.5%, 100%, preferably at least 80%, more preferably atleast 87%, even more preferably at least 90%, even more preferably atleast 95%, most preferably at least 97%, of the full-length of thepolynucleotide SEQ ID NO (or GenBank NO.) and retains a usable,functional characteristic (e.g., activity) of the full-lengthpolynucleotide molecule. As such, a fragment of a polynucleotide SEQ IDNO (or GenBank NO.) preferably means a nucleotide sequence thatcomprises or consists of the polynucleotide SEQ ID NO (or GenBank NO.),wherein an amount of consecutive nucleotides is missing and wherein theamount is no more than 50.0%, 40.0%, 30.0% of the full-length of thepolynucleotide SEQ ID NO (or GenBank NO.), preferably no more than20.0%, 15.0%, 10.0%, 9.0%, 8.0%, 7.0%, 6.0%, 5.0%, 4.5%, 4.0%, 3.5%,3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, more preferably no more than 15%,even more preferably no more than 10%, even more preferably no more than5%, most preferably no more than 2.5%, of the full-length of thepolynucleotide SEQ ID NO (or GenBank NO.) and wherein the fragmentretains a usable, functional characteristic (e.g., activity) of thefull-length polynucleotide molecule that can be routinely assessed bythe skilled person.

Throughout the application, the sequence of a polynucleotide can berepresented by a SEQ ID NO or alternatively GenBank NO. Therefore, theterms “polynucleotide SEQ ID NO” and “polynucleotide GenBank NO.” can beinterchangeably used, unless explicitly stated otherwise.

Fragments may additionally or alternatively include subsequences ofpolypeptides and protein molecules, or a subsequence of the polypeptide.In some cases, the fragment or domain is a subsequence of thepolypeptide that performs at least one biological function of the intactpolypeptide in substantially the same manner, preferably to a similarextent, as does the intact polypeptide. A “subsequence of thepolypeptide” as defined herein refers to a sequence of contiguous aminoacid residues derived from the polypeptide. For example, a polypeptidefragment can comprise a recognizable structural motif or functionaldomain such as a DNA-binding site or domain that binds to a DNA promoterregion, an activation domain, or a domain for protein-proteininteractions, and may initiate transcription. Fragments can vary in sizefrom as few as 3 amino acid residues to the full length of the intactpolypeptide, for example, at least about 20 amino acid residues inlength, for example, at least about 30 amino acid residues in length. Assuch, a fragment of a polypeptide SEQ ID NO (or UniProt ID or GenBankNO.) preferably means a polypeptide sequence that comprises or consistsof the polypeptide SEQ ID NO (or UniProt ID or GenBank NO.) wherein nomore than 80, 60, 50, 40, 30, 20 or 15 consecutive amino acid residuesare missing, preferably no more than 40 consecutive amino acid residuesare missing, and performs at least one biological function of the intactpolypeptide in substantially the same manner, preferably to a similar orgreater extent, as does the intact polypeptide that can be routinelyassessed by the skilled person. Alternatively, a fragment of apolypeptide SEQ ID NO (or UniProt ID or GenBank NO.) preferably means apolypeptide sequence that comprises or consists of an amount ofconsecutive amino acid residues from the polypeptide SEQ ID NO (orUniProt ID or GenBank NO.) and wherein the amount of consecutive aminoacid residues is at least 50.0%, 60.0%, 70.0%, 80.0%, 81.0%, 82.0%,83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%,93.0%, 94.0%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%,99.0%, 99.5%, 100%, preferably at least 80%, more preferably at least87%, even more preferably at least 90%, even more preferably at least95%, most preferably at least 97% of the full-length of the polypeptideSEQ ID NO (or UniProt ID or GenBank NO.) and that performs at least onebiological function of the intact polypeptide in substantially the samemanner, preferably to a similar or greater extent, as does the intactpolypeptide that can be routinely assessed by the skilled person. Assuch, a fragment of a polypeptide SEQ ID NO (or UniProt ID or GenBankNO.) preferably means a polypeptide sequence that comprises or consistsof the polypeptide SEQ ID NO (or UniProt ID or GenBank NO.), wherein anamount of consecutive amino acid residues is missing and wherein theamount is no more than 50.0%, 40.0%, 30.0% of the full-length of thepolypeptide SEQ ID NO (or UniProt ID or GenBank NO.), preferably no morethan 20.0%, 15.0%, 10.0%, 9.0%, 8.0%, 7.0%, 6.0%, 5.0%, 4.5%, 4.0%,3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, more preferably no more than15.0%, even more preferably no more than 10.0%, even more preferably nomore than 5.0%, most preferably no more than 2.5%, of the full-length ofthe polypeptide SEQ ID NO (or UniProt ID or GenBank NO.) and thatperforms at least one biological function of the intact polypeptide insubstantially the same manner, preferably to a similar or greaterextent, as does the intact polypeptide that can be routinely assessed bythe skilled person.

Throughout the application, the sequence of a polypeptide can berepresented by a SEQ ID NO or alternatively by a UniProt ID or GenBankNo. Therefore, the terms “polypeptide SEQ ID NO” and “polypeptideUniProt ID” and “polypeptide GenBank NO.” can be interchangeably used,unless explicitly stated otherwise.

Preferentially, a fragment of a polypeptide is a functional fragmentthat has at least one property or activity of the polypeptide from whichit is derived, preferably to a similar or greater extent. A functionalfragment can, for example, include a functional domain or conserveddomain of a polypeptide. It is understood that a polypeptide or afragment thereof may have conservative amino acid substitutions thathave substantially no effect on the polypeptide's activity. By“conservative substitutions” is intended substitution of one hydrophobicamino acid for another or substitution of one polar amino acid foranother or substitution of one acidic amino acid for another orsubstitution of one basic amino acid for another etc. Preferably, byconservative substitutions is intended combinations such as glycine byalanine and vice versa; valine, isoleucine and leucine by methionine andvice versa; aspartate by glutamate and vice versa; asparagine byglutamine and vice versa; serine by threonine and vice versa; lysine byarginine and vice versa; cysteine by methionine and vice versa; andphenylalanine and tyrosine by tryptophan and vice versa. A domain can becharacterized, for example, by a Pfam (El-Gebali et al., Nucleic AcidsRes. 47 (2019) D427-D432) or Conserved Domain Database (CDD)(ncbi.nlm.nih.gov/cdd) (Lu et al., Nucleic Acids Res. 48 (2020)D265-D268) designation. The content of each database is fixed at eachrelease and is not to be changed. When the content of a specificdatabase is changed, this specific database receives a new releaseversion with a new release date. All release versions for each databasewith their corresponding release dates and specific content as annotatedat these specific release dates are available and known to those skilledin the art. The PFAM database (pfam.xfam.org/) used herein was Pfamversion 33.1 released on Jun. 11, 2020. Protein sequence information andfunctional information can be provided by a comprehensive resource forprotein sequence and annotation data like, e.g., the Universal ProteinResource (UniProt) (uniprot.org) (Nucleic Acids Res. 2021, 49(D1),D480-D489). UniProt comprises the expertly and richly curated proteindatabase called the UniProt Knowledgebase (UniProtKB), together with theUniProt Reference Clusters (UniRef) and the UniProt Archive (UniParc).The UniProt identifiers (UniProt ID) are unique for each protein presentin the database. UniProt IDs as used herein are the UniProt IDs in theUniProt database version of 5 May 2021. Proteins that do not have aUniProt ID are referred herein using the respective GenBank Accessionnumber (GenBank No.) as present in the NIH genetic sequence database(ncbi.nlm.nih.gov/genbank/) (Nucleic Acids Res. 2013, 41(D1), D36-D42)version of 5 May 2021.

In the disclosure, polypeptide sequence stretches are being used torefer to fragments of the galactosyltransferases used in the disclosurethat are common to those galactosyltransferases. Such polypeptidestretches are written in the form of a sequence of amino acids inone-letter code. In case an amino acid at a specific place in suchpolypeptide stretch can be several amino acids, that specific place willhave amino acid code X.

Unless otherwise mentioned herein, the letter “X” refers to any aminoacid possible. The term (Xn) refers to a stretch of a protein sequenceconsisting of a number n of the amino acid residue X wherein each X isany amino acid possible and wherein n is 2, 3, 4 or more. The term (Xm)refers to a stretch of a protein sequence consisting of a number m ofthe amino acid residue X wherein each X is any amino acid possible andwherein m is 2, 3, 4 or more. The term (Xp) refers to a stretch of aprotein sequence consisting of a number p of the amino acid residue Xwherein each X is any amino acid possible and wherein p is 2, 3, 4 ormore.

The term “[X, no A, G or S]” refers to any amino acid excluding theamino acid residues alanine (A), glycine (G) or serine (S). The term“[X, no F, H, W or Y]” refers to any amino acid excluding the amino acidresidues phenylalanine (F), histidine (H), tryptophan (W) and tyrosine(Y). The term “[X, no V]” refers to any amino acid except for a valine(V).

The terms “nucleotide-sugar” or “activated sugar” as used herein referto activated forms of monosaccharides. Examples of activatedmonosaccharides include but are not limited to UDP-galactose (UDP-Gal),UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine(UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-fucose(GDP-Fuc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc),UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc orUDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,UDP-N-acetylfucosamine (UDP-L-FucNAc orUDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine(UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose),UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc orUDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose,CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid(CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N₃, CMP-Neu4,5Ac₂, CMP-Neu5,7Ac₂,CMP-Neu5,9Ac₂, CMP-Neu5,7(8,9)Ac₂, UDP-glucuronate, UDP-galacturonate,GDP-rhamnose, or UDP-xylose. Nucleotide-sugars act as glycosyl donors inglycosylation reactions. Those reactions are catalyzed by a group ofenzymes called glycosyltransferases.

The term “N-acetylglucosamine b-1,X-galactosyltransferase” as used inthe disclosure refers to an N-acetylglucosamineb-1,3-galactosyltransferase or an N-acetylglucosamineb-1,4-galactosyltransferase that transfers a galactosyl residue fromUDP-galactose to an acceptor in a beta-1,3 or beta-1,4 linkage,respectively.

The terms “N-acetylglucosamine b-1,3-galactosyltransferase,”“N-acetylglucosamine beta-1,3-galactosyltransferase,”“N-acetylglucosamine beta 1,3 galactosyltransferase,”“N-acetylglucosamine β-1,3-galactosyltransferase,” “N-acetylglucosamineβ1,3 galactosyltransferase” as used in the disclosure, are usedinterchangeably and refer to a galactosyltransferase that catalyzes thetransfer of galactose from the donor substrate UDP-galactose, to anacceptor in a beta-1,3 glycosidic linkage. A polynucleotide encoding an“N-acetylglucosamine b-1,3-galactosyltransferase” or any of the aboveterms, refers to a polynucleotide encoding such glycosyltransferase thatcatalyzes the transfer of galactose from the donor substrateUDP-galactose, to an acceptor in a beta-1,3 glycosidic linkage.

The terms “N-acetylglucosamine b-1,4-galactosyltransferase,”“N-acetylglucosamine beta-1,4-galactosyltransferase,”“N-acetylglucosamine beta 1,4 galactosyltransferase,”“N-acetylglucosamine β-1,4-galactosyltransferase,” “N-acetylglucosamineβ1,4 galactosyltransferase” as used in the disclosure, are usedinterchangeably and refer to a galactosyltransferase that catalyzes thetransfer of galactose from the donor substrate UDP-galactose, to anacceptor in a beta-1,4 glycosidic linkage. A polynucleotide encoding an“N-acetylglucosamine b-1,4-galactosyltransferase” or any of the aboveterms, refers to a polynucleotide encoding such glycosyltransferase thatcatalyzes the transfer of galactose from the donor substrateUDP-galactose, to an acceptor in a beta-1,4 glycosidic linkage.

The term “acceptor” as used herein refers to the monosaccharideN-acetylglucosamine (GlcNAc), the monosaccharide N-acetylgalactosamine(GalNAc), and/or an N-acetylglucosamine residue and/or anN-acetylgalactosamine residue as part of a di- and/or oligosaccharide atthe non-reducing end of the di- and/or oligosaccharide, that is modifiedby any one of N-acetylglucosamine b-1,X-galactosyltransferases of thedisclosure. Examples of the di- and/or oligosaccharides containing anN-acetylglucosamine and/or N-acetylgalactosamine at the non-reducing endinclude, but are not limited to, GlcNAc-b1,3-Glc, GlcNAc-b1,4-Glc,GalNAc-b1,3-Glc, GalNAc-b1,4-Glc, GlcNAc-b1,3-Gal-b1,4-Glc(lacto-N-triose, LN3), GalNAc-a1,3-Gal-b1,4-Glc (3′-GalNAcL),Gal-b1,4-GlcNAc-b1,6-(GlcNAc-b1,3)-Gal-b1,4-Glc,GalNAc-b1,3-Gal-b1,4-Glc (b3′-GalNAcL), GalNAc-b1,4-GlcNAc (LacdiNAc),GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, GalNAc-b1,4-Glc,Neu5Ac-a2,6-(GlcNAc-b1,3)-Gal-b1,4-Glc (6′-sialylated LN3),Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,6-(GlcNAc-b1,3)-Gal-b1,4-Glc,GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc,GalNAc-b1,4-(Neu5Ac-a2,3)-Gal-b1,4-Glc,GlcNAc-b1,6-(Gal-b1,3)-Gal-b1,4-Glc (Novo-LNT), lacto-N-pentaose (LNP),lacto-N-neopentaose, para lacto-N-pentaose, para lacto-N-neopentaose,lacto-N-novopentaose I, lacto-N-heptaose, lacto-N-neoheptaose, paralacto-N-neoheptaose, para lacto-N-heptaose, iso lacto-N-nonaose, novolacto-N-nonaose, lacto-N-nonaose.

The term “disaccharide” as used herein refers to a saccharide composedof two monosaccharide units. Examples of disaccharides comprise lactose(Gal-b1,4-Glc), lacto-N-biose (Gal-b1,3-GlcNAc), N-acetyllactosamine(Gal-b1,4-GlcNAc), LacDiNAc (GalNAc-b1,4-GlcNAc),N-acetylgalactosaminylglucose (GalNAc-b1,4-Glc), Neu5Ac-a2,3-Gal,Neu5Ac-a2,6-Gal and fucopyranosyl-(1-4)-N-glycolylneuraminic acid(Fuc-(1-4)-Neu5Gc).

“Oligosaccharide” as the term is used herein and as generally understoodin the state of the art, refers to a saccharide polymer containing asmall number, typically three to twenty, of simple sugars, i.e.,monosaccharides. The monosaccharides as used herein are reducing sugars.The disaccharides and oligosaccharides can be reducing or non-reducingsugars and have a reducing and a non-reducing end. A reducing sugar isany sugar that is capable of reducing another compound and is oxidizeditself, that is, the carbonyl carbon of the sugar is oxidized to acarboxyl group. The term “reducing end of a saccharide” as used in thedisclosure, refers to the free anomeric carbon that is available in thesaccharide to reduce another compound.

The term “monosaccharide” as used herein refers to a sugar that is notdecomposable into simpler sugars by hydrolysis, is classed either analdose or ketose, and contains one or more hydroxyl groups per molecule.Monosaccharides are saccharides containing only one simple sugar.Examples of monosaccharides comprise Hexose, D-Glucopyranose,D-Galactofuranose, D-Galactopyranose, L-Galactopyranose,D-Mannopyranose, D-Allopyranose, L-Altropyranose, D-Gulopyranose,L-Idopyranose, D-Talopyranose, D-Ribofuranose, D-Ribopyranose,D-Arabinofuranose, D-Arabinopyranose, L-Arabinofuranose,L-Arabinopyranose, D-Xylopyranose, D-Lyxopyranose, D-Erythrofuranose,D-Threofuranose, Heptose, L-glycero-D-manno-Heptopyranose (LDmanHep),D-glycero-D-manno-Heptopyranose (DDmanHep), 6-Deoxy-L-altropyranose,6-Deoxy-D-gulopyranose, 6-Deoxy-D-talopyranose,6-Deoxy-D-galactopyranose, 6-Deoxy-L-galactopyranose,6-Deoxy-D-mannopyranose, 6-Deoxy-L-mannopyranose,6-Deoxy-D-glucopyranose, 2-Deoxy-D-arabino-hexose,2-Deoxy-D-erythro-pentose, 2,6-Dideoxy-D-arabino-hexopyranose,3,6-Dideoxy-D-arabino-hexopyranose, 3,6-Dideoxy-L-arabino-hexopyranose,3,6-Dideoxy-D-xylo-hexopyranose, 3,6-Dideoxy-D-ribo-hexopyranose,2,6-Dideoxy-D-ribo-hexopyranose, 3,6-Dideoxy-L-xylo-hexopyranose,2-Amino-2-deoxy-D-glucopyranose, 2-Amino-2-deoxy-D-galactopyranose,2-Amino-2-deoxy-D-mannopyranose, 2-Amino-2-deoxy-D-allopyranose,2-Amino-2-deoxy-L-altropyranose, 2-Amino-2-deoxy-D-gulopyranose,2-Amino-2-deoxy-L-idopyranose, 2-Amino-2-deoxy-D-talopyranose,2-Acetamido-2-deoxy-D-glucopyranose,2-Acetamido-2-deoxy-D-galactopyranose,2-Acetamido-2-deoxy-D-mannopyranose, 2-Acetamido-2-deoxy-D-allopyranose,2-Acetamido-2-deoxy-L-altropyranose, 2-Acetamido-2-deoxy-D-gulopyranose,2-Acetamido-2-deoxy-L-idopyranose, 2-Acetamido-2-deoxy-D-talopyranose,2-Acetamido-2,6-dideoxy-D-galactopyranose,2-Acetamido-2,6-dideoxy-L-galactopyranose,2-Acetamido-2,6-dideoxy-L-mannopyranose,2-Acetamido-2,6-dideoxy-D-glucopyranose,2-Acetamido-2,6-dideoxy-L-altropyranose,2-Acetamido-2,6-dideoxy-D-talopyranose, D-Glucopyranuronic acid,D-Galactopyranuronic acid, D-Mannopyranuronic acid, D-Allopyranuronicacid, L-Altropyranuronic acid, D-Gulopyranuronic acid, L-Gulopyranuronicacid, L-Idopyranuronic acid, D-Talopyranuronic acid, Sialic acid,5-Amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid,5-Acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid,5-Glycolylamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid,Erythritol, Arabinitol, Xylitol, Ribitol, Glucitol, Galactitol,Mannitol, D-ribo-Hex-2-ulopyranose, D-arabino-Hex-2-ulofuranose(D-fructofuranose), D-arabino-Hex-2-ulopyranose,L-xylo-Hex-2-ulopyranose, D-lyxo-Hex-2-ulopyranose,D-threo-Pent-2-ulopyranose, D-altro-Hept-2-ulopyranose,3-C-(Hydroxymethyl)-D-erythofuranose,2,4,6-Trideoxy-2,4-diamino-D-glucopyranose,6-Deoxy-3-O-methyl-D-glucose, 3-O-Methyl-D-rhamnose,2,6-Dideoxy-3-methyl-D-ribo-hexose,2-Amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose,2-Acetamido-3-O-[(R)-carboxyethyl]-2-deoxy-D-glucopyranose,2-Glycolylamido-3-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose,3-Deoxy-D-lyxo-hept-2-ulopyranosaric acid,3-Deoxy-D-manno-oct-2-ulopyranosonic acid,3-Deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid,5,7-Diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulopyranosonicacid,5,7-Diamino-3,5,7,9-tetradeoxy-L-glycero-L-altro-non-2-ulopyranosonicacid,5,7-Diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulopyranosonicacid,5,7-Diamino-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulopyranosonicacid, glucose, galactose, N-acetylglucosamine, glucosamine, mannose,xylose, N-acetylmannosamine, N-acetylneuraminic acid,N-glycolylneuraminic acid, a sialic acid, N-acetylgalactosamine,galactosamine, fucose, rhamnose, glucuronic acid, gluconic acid,fructose and polyols.

With the term “polyol” is meant an alcohol containing multiple hydroxylgroups. For example, glycerol, sorbitol, or mannitol.

The term “galactosylated disaccharide” as used in the disclosurecomprises Gal-b1,3-GlcNAc, Gal-b1,4-GlcNAc, Gal-b1,3-GalNAc andGal-b1,4-GalNAc wherein galactose is linked to an N-acetylglucosamine(GlcNAc) or to an N-acetylgalactosamine (GalNAc), respectively, in abeta-1,3-linkage or a beta-1,4-linkage, and wherein theN-acetylglucosamine or N-acetylgalactosamine is positioned at thereducing end of the disaccharide and galactose at the terminalnon-reducing end of the disaccharide.

The terms “Gal-b1,3-GlcNAc,” “Gal-beta-1,3-GlcNAc,” “Gal-β1,3-GlcNAc,”“Galb1, 3GlcNAc,” “Galβ1,3GlcNAc,” “lacto-N-biose,” “LNB,” “LacNAc typeI,” “type 1 LacNAc,” “LacNAc (I)” are used interchangeably and refer toa disaccharide wherein galactose is linked to an N-acetylglucosamine ina beta-1,3-linkage, and wherein N-acetylglucosamine is positioned at thereducing end of the disaccharide.

The terms “Gal-b1,4-GlcNAc,” “Gal-beta-1,4-GlcNAc,” “Gal-β1,4-GlcNAc,”“Galb1,4GlcNAc,” “Galβ1,4GlcNAc,” “N-acetyllactosamine,” “LacNAc,”“LacNAc type II,” “type 2 LacNAc,” “LacNAc (II)” are usedinterchangeably and refer to a disaccharide wherein galactose is linkedto an N-acetylglucosamine in a beta-1,4-linkage, and whereinN-acetylglucosamine is positioned at the reducing end of thedisaccharide.

The terms “Gal-b1,3-GalNAc,” “Gal-beta-1,3-GalNAc,” “Gal-β1,3-GlcNAc,”“Galb1,3GalNAc,” “Galβ1,3GalNAc” and “T-disaccharide” are usedinterchangeably and refer to a disaccharide wherein galactose is linkedto an N-acetylgalactosamine in a beta-1,3-linkage, and whereinN-acetylgalactosamine is positioned at the reducing end of thedisaccharide.

The terms “Gal-b1,4-GalNAc,” “Gal-beta-1,4-GalNAc,” “Gal-β1,4-GalNAc,”“Galb1,4GalNAc,” “Galβ1,4GalNAc” are used interchangeably and refer to adisaccharide wherein galactose is linked to an N-acetylgalactosamine ina beta-1,4-linkage, and wherein N-acetylgalactosamine is positioned atthe reducing end of the disaccharide.

The term “galactosylated oligosaccharide” as used in the disclosurerefers to an oligosaccharide built of three to twenty monosaccharideunits, wherein a terminal non-reducing galactose is linked to anN-acetylglucosamine or an N-acetylgalactosamine of the oligosaccharidein a beta-1,3 or beta-1,4 linkage. The oligosaccharide as used in thedisclosure can be a linear structure or can include branches. Thelinkage (e.g., glycosidic linkage, galactosidic linkage, glucosidiclinkage, etc.) between two sugar units can be expressed, for example, as1,4, 1→4, or (1-4), used interchangeably herein. For example, the terms“Gal-b1,4-Glc,” “β-Gal-(1→4)-Glc,” “Galbeta1-4-Glc” and “Gal-b(1-4)-Glc”have the same meaning, i.e., a beta-glycosidic bond links carbon-1 ofgalactose (Gal) with the carbon-4 of glucose (Glc). Each monosaccharidecan be in the cyclic form (e.g., pyranose of furanose form). Linkagesbetween the individual monosaccharide units may include alpha 1→2, alpha1→3, alpha 1→4, alpha 1→6, alpha 2→1, alpha 2→3, alpha 2→4, alpha 2→6,beta 1→2, beta 1→3, beta 1→4, beta 1→6, beta 2→1, beta 2→3, beta 2→4,and beta 2→6. An oligosaccharide can contain both alpha- andbeta-glycosidic bonds or can contain only beta-glycosidic bonds.

The terms “glucosamine 6-phosphate N-acetyltransferase,”“glucosamine-phosphate N-acetyltransferase,” “GNA,” “GNA1,”“glucosamine-6P N-acetyltransferase,” “GlcN6P N-acetyltransferase” asused in the disclosure, are used interchangeably and refer to an enzymethat catalyzes the transfer of an acetyl group from acetyl-CoA to theprimary amine in glucosamine-6-phosphate, generatingN-acetyl-D-glucosamine-6-phosphate, the latter also known as GlcNAc-6P.A polynucleotide encoding a “glucosamine 6-phosphateN-acetyltransferase” or any of the above terms, refers to apolynucleotide encoding such an enzyme that catalyzes the transfer of anacetyl group from acetyl-CoA to the primary amine inglucosamine-6-phosphate, generating N-acetyl-D-glucosamine-6-phosphate.

The terms “fructose-6-phosphate aminotransferase,”“glutamine-fructose-6-phosphate-aminotransferase,”“glutamine-fructose-6-phosphate aminotransferase,” “L-glutamineD-fructose-6-phosphate aminotransferase,” “glmS,” “glms,” “glmS*54” asused in the disclosure, are used interchangeably and refer to an enzymethat catalyzes the conversion of fructose-6-phosphate intoglucosamine-6-phosphate using glutamine as a nitrogen source. Apolynucleotide encoding a “fructose-6-phosphate aminotransferase” or anyof the above terms, refers to a polynucleotide encoding such an enzymethat catalyzes the conversion of fructose-6-phosphate intoglucosamine-6-phosphate using glutamine as a nitrogen source.

The term “purified” refers to material that is substantially oressentially free from components that interfere with the activity of thebiological molecule. For cells, saccharides, nucleic acids, andpolypeptides, the term “purified” refers to material that issubstantially or essentially free from components that normallyaccompany the material as found in its native state. Typically, purifiedsaccharides, oligosaccharides, proteins or nucleic acids of thedisclosure are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85%pure, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% pure as measured by band intensity on a silver stained gelor other method for determining purity. Purity or homogeneity can beindicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a protein or nucleic acid sample,followed by visualization upon staining. For certain purposes highresolution will be needed and HPLC or a similar means for purificationutilized. For oligosaccharides, purity can be determined using methodssuch as but not limited to thin layer chromatography, gaschromatography, NMR, HPLC, capillary electrophoresis or massspectroscopy.

The terms “identical” or “percent identity” or “% identity” in thecontext of two or more nucleic acid or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame, when compared and aligned for maximum correspondence, as measuredusing sequence comparison algorithms or by visual inspection. Forsequence comparison, one sequence acts as a reference sequence, to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are inputted into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters. Percent identity may be calculated globally over thefull-length sequence of the reference sequence, resulting in a globalpercent identity score. Alternatively, percent identity may becalculated over a partial sequence of the reference sequence, resultingin a local percent identity score. Using the full-length of thereference sequence in a local sequence alignment results in a globalpercent identity score between the test and the reference sequence.Percent identity can be determined using different algorithms like, forexample, BLAST and PSI-BLAST (Altschul et al., 1990, J. Mol. Biol.215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res. 25: 17,3389-402), the Clustal Omega method (Sievers et al., 2011, Mol. Syst.Biol. 7:539), the MatGAT method (Campanella et al., 2003, BMCBioinformatics, 4:29) or EMBOSS Needle.

The BLAST (Basic Local Alignment Search Tool)) method of alignment is analgorithm provided by the National Center for Biotechnology Information(NCBI) to compare sequences using default parameters. The programcompares nucleotide or protein sequences to sequence databases andcalculates the statistical significance. PSI-BLAST (Position-SpecificIterative Basic Local Alignment Search Tool) derives a position-specificscoring matrix (PSSM) or profile from the multiple sequence alignment ofsequences detected above a given score threshold using protein-proteinBLAST (BLASTp). The BLAST method can be used for pairwise or multiplesequence alignments. Pairwise Sequence Alignment is used to identifyregions of similarity that may indicate functional, structural and/orevolutionary relationships between two biological sequences (protein ornucleic acid). The web interface for BLAST is available atblast.ncbi.nlm.nih.gov/Blast.cgi.

Clustal Omega (Clustal W) is a multiple sequence alignment program thatuses seeded guide trees and HMM profile-profile techniques to generatealignments between three or more sequences. It produces biologicallymeaningful multiple sequence alignments of divergent sequences. The webinterface for Clustal W is available at ebi.ac.uk/Tools/msa/clustalo/.Default parameters for multiple sequence alignments and calculation ofpercent identity of protein sequences using the Clustal W method are:enabling de-alignment of input sequences: FALSE; enabling mbed-likeclustering guide-tree: TRUE; enabling mbed-like clustering iteration:TRUE; Number of (combined guide-tree/IMM) iterations: default(0); MaxGuide Tree Iterations: default [−1]; Max HMM Iterations: default [−1];order: aligned.

MatGAT (Matrix Global Alignment Tool) is a computer application thatgenerates similarity/identity matrices for DNA or protein sequenceswithout needing pre-alignment of the data. The program performs a seriesof pairwise alignments using the Myers and Miller global alignmentalgorithm, calculates similarity and identity, and then places theresults in a distance matrix. The user may specify which type ofalignment matrix (e.g., BLOSUM50, BLOSUM62, and PAM250) to employ withtheir protein sequence examination.

EMBOSS Needle (galaxy-iuc.github.io/emboss-5.0-docs/needle.html) usesthe Needleman-Wunsch global alignment algorithm to find the optimalalignment (including gaps) of two sequences when considering theirentire length. The optimal alignment is ensured by dynamic programmingmethods by exploring all possible alignments and choosing the best. TheNeedleman-Wunsch algorithm is a member of the class of algorithms thatcan calculate the best score and alignment in the order of mn steps,(where “n” and “m” are the lengths of the two sequences). The gap openpenalty (default 10.0) is the score taken away when a gap is created.The default value assumes you are using the EBLOSUM62 matrix for proteinsequences. The gap extension (default 0.5) penalty is added to thestandard gap penalty for each base or residue in the gap. This is howlong gaps are penalized.

As used herein, a polypeptide having an amino acid sequence having atleast 80% sequence identity to the full-length sequence of a referencepolypeptide sequence is to be understood as that the sequence has 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.50%, 92.00%,92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95, 50%, 96.00%, 96,50%,97.00%, 97,50%, 98.00%, 98,50%, 99.00%, 99,50%, 99,60%, 99,70%, 99,80%,99,90%, 100% sequence identity to the full-length of the amino acidsequence of the reference polypeptide sequence. Throughout theapplication, unless explicitly specified otherwise, a polypeptide (orDNA sequence) comprising/consisting/having an amino acid sequence (ornucleotide sequence) having at least 80% sequence identity to thefull-length amino acid sequence (or nucleotide sequence) of a referencepolypeptide (or nucleotide sequence), usually indicated with a SEQ ID NOor UniProt ID or GenBank NO., preferably has at least 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, more preferably has at least85%, even more preferably has at least 90%, most preferably has at least95%, sequence identity to the full length reference sequence.

For the purposes of this disclosure, percent identity is determinedusing MatGAT2.01 (Campanella et al., 2003, BMC Bioinformatics 4:29). Thefollowing default parameters for protein are employed: (1) Gap costExistence: 12 and Extension: 2; (2) The Matrix employed was BLOSUM65. Ina preferred embodiment, sequence identity is calculated based on thefull-length sequence of a given SEQ ID NO, i.e., the reference sequence,or a part thereof. Part thereof preferably means at least 50%, 60%, 70%,80%, 90% or 95% of the complete reference sequence.

The term “cultivation” refers to the culture medium wherein the cell iscultivated or fermented, the cell itself, and the galactosylated di-and/or oligosaccharides that are produced by the cell of the disclosurein whole broth, i.e., inside (intracellularly) as well as outside(extracellularly) of the cell.

“Mammalian milk oligosaccharides (MMOs)” comprise oligosaccharidespresent in milk found in any phase during lactation including colostrummilk from humans (i.e., human milk oligosaccharides or HMOs) and mammalsincluding but not limited to cows (Bos Taurus), sheep (Ovis aries),goats (Capra aegagrus hircus), bactrian camels (Camelus bactrianus),horses (Equus ferus caballus), pigs (Sus scropha), dogs (Canis lupusfamiliaris), ezo brown bears (Ursus arctos yesoensis), polar bear (Ursusmaritimus), Japanese black bears (Ursus thibetanus japonicus), stripedskunks (Mephitis mephitis), hooded seals (Cystophora cristata), Asianelephants (Elephas maximus), African elephant (Loxodonta africana),giant anteater (Myrmecophaga tridactyla), common bottlenose dolphins(Tursiops truncates), northern minke whales (Balaenopteraacutorostrata), tammar wallabies (Macropus eugenii), red kangaroos(Macropus rufus), common brushtail possum (Trichosurus Vulpecula),koalas (Phascolarctos cinereus), eastern quolls (Dasyurus viverrinus),platypus (Ornithorhynchus anatinus). Human milk oligosaccharides (HMOs)are also known as human identical milk oligosaccharides that arechemically identical to the human milk oligosaccharides found in humanbreast milk but that are biotechnologically-produced (e.g., using cellfree systems or cells and organisms comprising a bacterium, a fungus, ayeast, a plant, animal, or protozoan cell, preferably geneticallyengineered cells and organisms). Human identical milk oligosaccharidesare marketed under the name HiMO.

The term “membrane transporter proteins” as used herein refers toproteins that are part of or interact with the cell membrane and controlthe flow of molecules and information across the cell. The membraneproteins are thus involved in transport, be it import into or export outof the cell.

Such membrane transporter proteins can be porters,P—P-bond-hydrolysis-driven transporters, β-Barrel Porins, auxiliarytransport proteins, putative transport proteins andphosphotransfer-driven group translocators as defined by the TransporterClassification Database that is operated and curated by the Saier LabBioinformatics Group available via tcdb.org and providing a functionaland phylogenetic classification of membrane transport proteins ThisTransporter Classification Database details a comprehensive IUBMBapproved classification system for membrane transporter proteins knownas the Transporter Classification (TC) system. The TCDB classificationsearches as described here are defined based on TCDB.org as released on17^(th) June 2019.

Porters is the collective name of uniporters, symporters, andantiporters that utilize a carrier-mediated process (Saier et al.,Nucleic Acids Res. 44 (2016) D372-D379). They belong to theelectrochemical potential-driven transporters and are also known assecondary carrier-type facilitators. Membrane transporter proteins areincluded in this class when they utilize a carrier-mediated process tocatalyze uniport when a single species is transported either byfacilitated diffusion or in a membrane potential-dependent process ifthe solute is charged; antiport when two or more species are transportedin opposite directions in a tightly coupled process, not coupled to adirect form of energy other than chemiosmotic energy; and/or symportwhen two or more species are transported together in the same directionin a tightly coupled process, not coupled to a direct form of energyother than chemiosmotic energy, of secondary carriers (Forrest et al.,Biochim. Biophys. Acta. 1807 (2011) 167-188). These systems are usuallystereospecific. Solute:solute countertransport is a characteristicfeature of secondary carriers. The dynamic association of porters andenzymes creates functional membrane transport metabolons that channelsubstrates typically obtained from the extracellular compartmentdirectly into their cellular metabolism (Moraes and Reithmeier, Biochim.Biophys. Acta. 1818 (2012), 2687-2706). Solutes that are transported viathis porter system include but are not limited to cations, organicanions, inorganic anions, nucleosides, amino acids, polyols,phosphorylated glycolytic intermediates, osmolytes, and siderophores.

Membrane transporter proteins are included in the class of P—P-bondhydrolysis-driven transporters if they hydrolyze the diphosphate bond ofinorganic pyrophosphate, ATP, or another nucleoside triphosphate, todrive the active uptake and/or extrusion of a solute or solutes (Saieret al., Nucleic Acids Res. 44 (2016) D372-D379). The membranetransporter protein may or may not be transiently phosphorylated, butthe substrate is not phosphorylated. Substrates that are transportedvithe class of P—P-bond hydrolysis-driven transporters include but arenot limited to cations, heavy metals, beta-glucan, UDP-glucose,lipopolysaccharides, teichoic acid.

The β-Barrel porins membrane transporter proteins form transmembranepores that usually allow the energy independent passage of solutesacross a membrane. The transmembrane portions of these proteins consistexclusively of β-strands that form a β-barrel (Saier et al., NucleicAcids Res. 44 (2016) D372-D379). These porin-type proteins are found inthe outer membranes of Gram-negative bacteria, mitochondria, plastids,and possibly acid-fast Gram-positive bacteria. Solutes that aretransported vithese β-Barrel porins include but are not limited tonucleosides, raffinose, glucose, beta-glucosides, and oligosaccharides.

The auxiliary transport proteins are defined to be proteins thatfacilitate transport across one or more biological membranes but do notthemselves participate directly in transport. These membrane transporterproteins always function in conjunction with one or more establishedtransport systems such as but not limited to outer membrane factors(OMFs), polysaccharide (PST) porters, the ATP-binding cassette(ABC)-type transporters. They may provide a function connected withenergy coupling to transport, play a structural role in complexformation, serve a biogenic or stability function or function inregulation (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379).Examples of auxiliary transport proteins include but are not limited tothe polysaccharide copolymerase family involved in polysaccharidetransport, the membrane fusion protein family involved in bacteriocinand chemical toxin transport.

Putative transport protein comprises families that will either beclassified elsewhere when the transport function of a member becomesestablished or will be eliminated from the Transporter Classificationsystem if the proposed transport function is disproven. These familiesinclude a member or members for which a transport function has beensuggested, but evidence for such a function is not yet compelling (Saieret al., Nucleic Acids Res. 44 (2016) D372-D379). Examples of putativetransporters classified in this group under the TCDB system as releasedon 17^(th) June 2019 include but are not limited to copper transporters.

The phosphotransfer-driven group translocators are also known as thePEP-dependent phosphoryl transfer-driven group translocators of thebacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). Theproduct of the reaction, derived from extracellular sugar, is acytoplasmic sugar-phosphate. The enzymatic constituents, catalyzingsugar phosphorylation, are superimposed on the transport process in atightly coupled process. The PTS system is involved in many differentaspects comprising in regulation and chemotaxis, biofilm formation, andpathogenesis (Lengeler, J. Mol. Microbiol. Biotechnol. 25 (2015) 79-93;Saier, J. Mol. Microbiol. Biotechnol. 25 (2015) 73-78). Membranetransporter protein families classified within thephosphotransfer-driven group translocators under the TCDB system asreleased on Jun. 17, 2019, include PTS systems linked to transport ofglucose-glucosides, fructose-mannitol,lactose-N,N′-diacetylchitobiose-beta-glucoside, glucitol, galactitol,mannose-fructose-sorbose and ascorbate.

The major facilitator superfamily (MFS) is a superfamily of membranetransporter proteins catalyzing uniport, solute:cation (H+, but seldomNa+) symport and/or solute:H+ or solute:solute antiport. Most are of400-600 amino acyl residues in length and possess either 12, 14, oroccasionally, 24 transmembrane α-helical spanners (TMSs) as defined bythe Transporter Classification Database operated by the Saier LabBioinformatics Group (tcdb.org).

“SET” or “Sugar Efflux Transporter” as used herein refers to membraneproteins of the SET family that are proteins with InterPro domainIPR004750 and/or are proteins that belong to the eggNOGv4.5 familyENOG410XTE9. Identification of the InterPro domain can be done by usingthe online tool on https://www.ebi.ac.uk/interpro/or a standaloneversion of InterProScan (ebi.ac.uk/interpro/download.html) using thedefault values. Identification of the orthology family in eggNOGv4.5 canbe done using the online version or a standalone version ofeggNOG-mapperv1 (eggnogdb.embl.de/#/app/home).

The term “Siderophore,” as used herein, is referring to the secondarymetabolite of various microorganisms that are mainly ferric ion specificchelators. These molecules have been classified as catecholate,hydroxamate, carboxylate and mixed types. Siderophores are in generalsynthesized by a nonribosomal peptide synthetase (NRPS) dependentpathway or an NRPS independent pathway (NIS). The most importantprecursor in NRPS-dependent siderophore biosynthetic pathway ischorismate. 2,3-DHBA could be formed from chorismate by a three-stepreaction catalyzed by isochorismate synthase, isochorismatase, and2,3-dihydroxybenzoate-2,3-dehydrogenase. Siderophores can also be formedfrom salicylate, which is formed from isochorismate by isochorismatepyruvate lyase. When ornithine is used as precursor for siderophores,biosynthesis depends on the hydroxylation of ornithine catalyzed byL-ornithine N5-monooxygenase. In the NIS pathway, an important step insiderophore biosynthesis is N(6)-hydroxylysine synthase.

A transporter is needed to export the siderophore outside the cell. Foursuperfamilies of membrane proteins are identified so far in thisprocess: the major facilitator superfamily (MFS); theMultidrug/Oligosaccharidyl-lipid/Polysaccharide Flippase Superfamily(MOP); the resistance, nodulation and cell division superfamily (RND);and the ABC superfamily. In general, the genes involved in siderophoreexport are clustered together with the siderophore biosynthesis genes.The term “siderophore exporter” as used herein refers to suchtransporters needed to export the siderophore outside of the cell.

The ATP-binding cassette (ABC) superfamily contains both uptake andefflux transport systems, and the members of these two groups generallycluster loosely together. ATP hydrolysis without protein phosphorylationenergizes transport. There are dozens of families within the ABCsuperfamily, and family generally correlates with substrate specificity.Members are classified according to class 3.A.1 as defined by theTransporter Classification Database operated by the Saier LabBioinformatics Group available via www.tcdb.org and providing afunctional and phylogenetic classification of membrane transporterproteins.

The term “enabled efflux” means to introduce the activity of transportof a solute over the cytoplasm membrane and/or the cell wall. Thetransport may be enabled by introducing and/or increasing the expressionof a transporter protein as described in the disclosure. The term“enhanced efflux” means to improve the activity of transport of a soluteover the cytoplasm membrane and/or the cell wall. Transport of a soluteover the cytoplasm membrane and/or cell wall may be enhanced byintroducing and/or increasing the expression of a membrane transporterprotein as described in the disclosure. “Expression” of a membranetransporter protein is defined as “overexpression” of the gene encodingthe membrane transporter protein in the case the gene is an endogenousgene or “expression” in the case the gene encoding the membranetransporter protein is a heterologous gene that is not present in thewild type strain or cell.

The term “precursor” as used herein refers to substances that are takenup and/or synthetized by the cell for the specific production of anoligosaccharide. In this sense a precursor can be an acceptor as definedherein, but can also be another substance, metabolite, which is firstmodified within the cell as part of the biochemical synthesis route ofthe oligosaccharide. Examples of such precursors comprise the acceptorsas defined herein, and/or glucose, galactose, fructose, glycerol, sialicacid, fucose, mannose, maltose, sucrose, lactose, dihydroxyacetone,glucosamine, N-acetyl-glucosamine, mannosamine, N-acetyl-mannosamine,galactosamine, N-acetylgalactosamine, phosphorylated sugars like, e.g.,but not limited to glucose-1-phosphate, galactose-1-phosphate,glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate,mannose-6-phosphate, mannose-1-phosphate, glycerol-3-phosphate,glyceraldehyde-3-phosphate, dihydroxyacetone-phosphate,glucosamine-6-phosphate, N-acetyl-glucosamine-6-phosphate,N-acetylmannosamine-6-phosphate, N-acetylglucosamine-1-phosphate,N-acetyl-neuraminic acid-9-phosphate and/or nucleotide-activated sugarsas defined herein like, e.g., UDP-glucose, UDP-galactose,UDP-N-acetylglucosamine, CMP-sialic acid, GDP-mannose,GDP-4-dehydro-6-deoxy-α-D-mannose, and GDP-fucose.

Throughout the application, unless explicitly stated otherwise, thefeatures “synthesize,” “synthesized” and “synthesis” are interchangeablyused with the features “produce,” “produced” and “production,”respectively.

Description

According to a first aspect, the disclosure provides the use of anN-acetylglucosamine b-1,X-galactosyltransferase for the synthesis of agalactosylated disaccharide or oligosaccharide. Herein,N-acetylglucosamine b-1,X-galactosyltransferase galactosylates anN-acetylglucosamine and/or N-acetylgalactosamine as a monosaccharide,and/or an N-acetylglucosamine and/or N-acetylgalactosamine as part of adi- and/or oligosaccharide at the non-reducing end of the di- and/oroligosaccharide to produce the galactosylated disaccharide oroligosaccharide. Throughout the application, the feature “di- oroligosaccharide” is preferably replaced with “oligosaccharide,” thefeature “di- and/or oligosaccharides” is preferably replaced with“oligosaccharides.” Throughout the application, the di- and/oroligosaccharide is preferably a mammalian milk oligosaccharide (MMO),more preferably a human milk oligosaccharide (HMO).

In the scope of the disclosure, the N-acetylglucosamineb-1,X-galactosyltransferase is used to transfer a galactose residue fromthe nucleotide-sugar donor UDP-galactose to an N-acetylglucosamineand/or N-acetylgalactosamine as a monosaccharide, and/or anN-acetylglucosamine and/or N-acetylgalactosamine as part of a di- and/oroligosaccharide at the non-reducing end of the di- and/oroligosaccharide resulting in the production of a galactosylateddisaccharide or oligosaccharide as defined herein.

According to the disclosure, the N-acetylglucosamineb-1,X-galactosyltransferase is an N-acetylglucosamineb-1,3-galactosyltransferase or an N-acetylglucosarnineb-1,4-galactosyltransferase that transfers a galactose residue fromUDP-galactose to an N-acetylglucosamine and/or N-acetylgalactosamine asa monosaccharide, and/or an N-acetylglucosamine and/orN-acetylgalactosamine as part of a di- and/or oligosaccharide at thenon-reducing end of the di- and/or oligosaccharide in a beta-1,3 orbeta-1,4 linkage, respectively, resulting in the production of agalactosylated disaccharide or oligosaccharide as defined herein.

A galactosylated disaccharide according to the disclosure is asaccharide of two monosaccharides consisting of a galactose residue atits non-reducing end, which is beta-1,3 or beta-1,4 linked to a GleNAcor a GalNAc residue at its reducing end, and a galactosylatedoligosaccharide is a saccharide of three or more monosaccharides havinga terminal galactose residue at its non-reducing end, which is beta-1,3or beta-1,4 linked to a GlcNAc or a GalNAc residue.

According to the disclosure, the N-acetylglucosamineb-1,X-galactosyltransferase is an N-acetylglucosamineb-1,3-galactosyltransferase that has 1) PFAM domain PF00535 and that (i)comprises the sequence [AGPS]XXLN(Xn)RXDXD with SEQ ID NO: 1, wherein Xis any amino acid except for the combination XX on positions 2 and 3that cannot be an FA, FS, YC or YS combination and wherein n is 12 to17, or (ii) comprises the sequencePXXLN(Xn)RXDXD(Xm)[FWY]XX[HKR]XX[NQST] with SEQ ID NO: 2, wherein X isany amino acid except for the combination XX on positions 2 and 3 thatcannot be an FA, FS, YC or YS combination and wherein n is 12 to 17 andm is 100 to 115, or (iii) comprises a polypeptide sequence according toany one of SEQ ID NOs: 3 or 4, or (iv) is a functional homologue,variant or derivative of any one of SEQ ID NOs: 3 or 4 having at least80% overall sequence identity to the full length of any one of theN-acetylglucosamine b-1,3-galactosyltransferase polypeptide with SEQ IDNOs: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferaseactivity, or (v) comprises an oligopeptide sequence of at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acidresidues from any one of SEQ ID NOs: 3 or 4 and havingN-acetylglucosamine b-1,3-galactosyltransferase activity, or (vi) is afunctional fragment of any one of SEQ ID NOs: 3 or 4 and havingN-acetylglucosamine b-1,3-galactosyltransferase activity, or (vii)comprises a polypeptide comprising or consisting of an amino acidsequence having at least 80% sequence identity to the full-length aminoacid sequence of any one of SEQ ID NOs: 3 or 4 and havingN-acetylglucosamine b-1,3-galactosyltransferase activity; or theN-acetylglucosamine b-1,X-galactosyltransferase is anN-acetylglucosamine b-1,3-galactosyltransferase that has 2) PFAM domainIPR002659 and that (i) comprises the sequenceKT(Xn)[FY]XXKXDXD(Xm)[FHY]XXG(X, no A, G, S)(Xp)(X, no F, H, W,Y)[DE]D[ILV]XX[AG] with SEQ ID NO: 05, wherein X is any amino acid andwherein n is 13 to 16, m is 35 to 70 and p is 20 to 45, or (ii)comprises a polypeptide sequence according to any one of SEQ ID NOs: 6,7, 8 or 9, or (iii) is a functional homologue, variant or derivative ofany one of SEQ ID NOs: 6, 7, 8 or 9 having at least 80% overall sequenceidentity to the full length of any one of the N-acetylglucosamineb-1,3-galactosyltransferase polypeptide with SEQ ID NOs: 6, 7, 8 or 9and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or(iv) comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from anyone of SEQ ID NOs: 6, 7, 8 or 9 and having N-acetylglucosamineb-1,3-galactosyltransferase activity, or (v) is a functional fragment ofany one of SEQ ID NOs: 6, 7, 8 or 9 and having N-acetylglucosamineb-1,3-galactosyltransferase activity, or (vi) comprises a polypeptidecomprising or consisting of an amino acid sequence having at least 80%sequence identity to the full-length amino acid sequence of any one ofSEQ ID NOs: 6, 7, 8 or 9 and having N-acetylglucosamineb-1,3-galactosyltransferase activity. In a preferred embodiment, theN-acetylglucosamine b-1,3-galactosyltransferase as disclosed herein isnot beta1,3-galactosyltransferase (WbgO) from E. coli 055:H7 withUniProt ID D3QY14.

Alternatively, the N-acetylglucosamine b-1,X-galactosyltransferase is anN-acetylglucosamine b-1,4-galactosyltransferase that has 1) PFAM domainPF01755 and that (i) comprises the sequenceEXXCXXSHX[AFIL,TY]LW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 10,wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75,or (ii) comprises the sequenceEXXCXXSH[LR]VLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 11, wherein Xis any amino acid and wherein n is 13 to 15 and m is 50 to 75, or (iii)comprises the sequence EXXCXXSH[VHI]SLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] withSEQ ID NO: 12, wherein X is any amino acid and wherein n is 13 to 15 andm is 50 to 75, or (iv) comprises the sequenceEXXCXXSHYMLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 13, wherein X isany amino acid and wherein n is 13 to 15 and m is 50 to 75, or (v)comprises the sequence EXXCXXSHXX(X, no V)Y(Xn)EDD(Xm)[ACGST]XXY[ILMV]with SEQ ID NO: 14, wherein X is any amino acid and wherein n is 13 to15 and m is 50 to 75, or (vi) comprises a polypeptide sequence accordingto any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23, or (vii)is a functional homologue, variant or derivative of any one of SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23 having at least 80% overallsequence identity to the full length of any one of theN-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or (viii) comprises anoligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22 or 23 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or (ix) is a functional fragmentof any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23 andhaving N-acetylglucosamine b-1,4-galactosyltransferase activity, or (x)comprises a polypeptide comprising or consisting of an amino acidsequence having at least 80% sequence identity to the full-length aminoacid sequence of any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22or 23 and having N-acetylglucosamine b-1,4-galactosyltransferaseactivity; or the N-acetylglucosamine b-1,X-galactosyltransferase is anN-acetylglucosamine b-1,4-galactosyltransferase that has 2) PFAM domainPF00535 and that (i) comprises the sequence R[KN]XXXXXXXGXXXX[FL](X, noV)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE] with SEQ ID NO: 24 wherein X is anyamino acid and wherein n is 50 to 75 and m is 10 to 30, or (ii)comprises the sequence R[KN]XXXXXXXGXXXX[FL](X, noV)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE](Xp)[FWY]XX[HKR]XX[NQST] with SEQ ID NO:25 wherein X is any amino acid and wherein n is 50 to 75, m is 10 to 30and p is 20 to 25, or (iii) comprises a polypeptide sequence accordingto any one of SEQ ID NOs: 26 or 27, or (iv) is a functional homologue,variant or derivative of any one of SEQ ID NOs: 26 or 27 having at least80% overall sequence identity to the full length of any one of theN-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ IDNOs: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferaseactivity, or (v) comprises an oligopeptide sequence of at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acidresidues from any one of SEQ ID NOs: 26 or 27 and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, or (vi) is afunctional fragment of any one of SEQ ID NOs: 26 or 27 and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, or (vii)comprises a polypeptide comprising or consisting of an amino acidsequence having at least 80% sequence identity to the full-length aminoacid sequence of any one of SEQ ID NOs: 26 or 27 and havingN-acetylglucosamine b-1,4-galactosyltransferase activity; or theN-acetylglucosamine b-1,X-galactosyltransferase is anN-acetylglucosamine b-1,4-galactosyltransferase that has 3) PFAM domainPF02709 and not PFAM domain PF00535 and that (i) comprises the sequence[FWY]XX[FWY](Xn)[FWY][GQ]X[DE]D with SEQ ID NO: 28 wherein X is anyamino acid except for the combination XX on positions 2 and 3 thatcannot be an IP or NL combination and wherein n is 21 to 26, or (ii)comprises a polypeptide sequence according to any one of SEQ ID NOs: 29,30, 31, 32, 33 or 34, or (iii) is a functional homologue, variant orderivative of any one of SEQ ID NOs: 29, 30, 31, 32, 33 or 34 having atleast 80% overall sequence identity to the full length of any one of theN-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ IDNOs: 29, 30, 31, 32, 33 or 34 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or (iv) comprises an oligopeptidesequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20consecutive amino acid residues from any one of SEQ ID NOs: 29, 30, 31,32, 33 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferaseactivity, or (v) is a functional fragment of any one of SEQ ID NOs: 29,30, 31, 32, 33 or 34 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or (vi) comprises a polypeptidecomprising or consisting of an amino acid sequence having at least 80%sequence identity to the full-length amino acid sequence of any one ofSEQ ID NOs: 29, 30, 31, 32, 33 or 34 and having N-acetylglucosamineb-1,4-galactosyltransferase activity; or the N-acetylglucosamineb-1,X-galactosyltransferase is an N-acetylglucosamineb-1,4-galactosyltransferase that has 4) PFAM domain PF03808 and that (i)comprises the sequence[ST][FHY]XN(Xn)DGXXXXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA with SEQ ID NO:35, wherein X is any amino acid and wherein n is 20 to 25, or (ii)comprises the sequence[ST][FHY]XN(Xn)DGXXXXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA(Xm)[HR]XG[FWY](Xp)GXGXXXQ[DE] with SEQ ID NO: 36, wherein X is any aminoacid and wherein n is 20 to 25, m is 40 to 50 and p is 22 to 30, or(iii) comprises a polypeptide sequence according to any one of SEQ IDNOs: 37, 38 or 39, or (iv) is a functional homologue, variant orderivative of any one of SEQ ID NOs: 37, 38 or 39 having at least 80%overall sequence identity to the full length of any one of theN-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ IDNOs: 37, 38 or 39 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or (v) comprises an oligopeptidesequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20consecutive amino acid residues from any one of SEQ ID NOs: 37, 38 or 39and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or(vi) is a functional fragment of any one of SEQ ID NOs: 37, 38 or 39 andhaving N-acetylglucosamine b-1,4-galactosyltransferase activity, or(vii) comprises a polypeptide comprising or consisting of an amino acidsequence having at least 80% sequence identity to the full-length aminoacid sequence of any one of SEQ ID NOs: 37, 38 or 39 and havingN-acetylglucosamine b-1,4-galactosyltransferase activity. In a preferredembodiment, the N-acetylglucosamine b-1,4-galactosyltransferase asdisclosed herein is not Neisseria meningitidis 1gtB (UniProt ID Q51116).

The PFAM domains used herein, PF00535, IPR002659, PF01755, PF02709,PF03808, are protein domains as annotated in the PFAM database versionPfam 33.1 as released on Jun. 11, 2020. PF00535 is found in theglycosyltransferase 2 (GT2) family that comprises enzymes that transfersugar from UDP-glucose, UDP-N-acetyl-galactosamine, GDP-mannose orCDP-abequose, to a range of substrates including cellulose, dolicholphosphate and teichoic acids.

IPR002659 is found in the glycosyltransferase family 31 (GH31) thatcomprises enzymes with a number of known activities includingN-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase(2.4.1.149), beta-1,3-galactosyltransferase (2.4.1), fucose-specificbeta-1,3-N-acetylglucosaminyltransferase (2.4.1), globotriosylceramidebeta-1,3-GalNAc transferase (2.4.1.79). PF01755 is found in theglycosyltransferase 25 (GT25) family. This is a family ofglycosyltransferases involved in lipopolysaccharide (LPS) biosynthesis.These enzymes catalyze the transfer of various sugars onto the growingLPS chain during its biosynthesis. PF02709 refers to the Glyco_transf_7Cfamily. This is the N-terminal domain of a family ofgalactosyltransferases from a wide range of Metazoa with three relatedgalactosyltransferases activities, all three of which are possessed byone sequence in some cases: EC:2.4.1.90, N-acetyllactosamine synthase,EC:2.4.1.38, Beta-N-acetylglucosaminyl-glycopeptidebeta-1,4-galactosyltransferase, and EC:2.4.1.22 Lactose synthase.PF03808 refers to the glycosyltransferase 26 (GT26) family thatcomprises enzymes with activities like β-N-acetylmannosaminuronyltransferase (EC 2.4.1.-),β-N-acetyl-mannosaminyltransferase (EC 2.4.1.-),β-1,4-glucosyltransferase (EC 2.4.1.-) and β-1,4-galactosyltransferase(EC 2.4.1.-).

Proteins having the same PFAM domain and motifs as specified for eachclass of N-acetylglucosamine b-1,3-galactosyltransferase orN-acetylglucosamine b-1,4-galactosyltransferase can be searched via aRegEx analysis as exemplified in the disclosure. A RegEx, or RegularExpression, is a special sequence of characters that helps to match orfind other strings or sets of strings, using a specialized syntax heldin a pattern. Many programs are available to do RegEx search. One ofthem is the Python module “re,” which provides full support for regularexpressions in Python. Detailed information, and known by the personsskilled in the art, is available fromhttps://towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2,as released on 6 Apr. 2019.

The overall sequence identity may be determined using a global alignmentalgorithm, such as the Needleman Wunsch algorithm in the program GAP(GCG Wisconsin Package, Accelrys), preferably with default parametersand preferably with sequences of mature proteins (i.e., without takinginto account secretion signals or transit peptides). Compared to overallsequence identity, the sequence identity will generally be higher whenonly conserved domains or motifs are considered.

At least 80% overall sequence identity to the full length of any one ofthe polypeptides with SEQ ID NOs: 3, 4, 6, 7, 8, 9, 15, 16, 17, 18, 19,20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 or 39 should beunderstood as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequenceidentity to any one of the polypeptides with SEQ ID NOs: 3, 4, 6, 7, 8,9, 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34,37, 38 or 39, respectively, as given herein. An oligopeptide sequence ofat least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutiveamino acid residues from any one of the polypeptides with SEQ ID NOs: 3,4, 6, 7, 8, 9, 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31,32, 33, 34, 37, 38 or 39 should be understood as any one of oligopeptidesequences of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20up to the total number of amino acid residues, of consecutive amino acidresidues from any one of the polypeptides with SEQ ID NOs: 3, 4, 6, 7,8, 9, 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33,34, 37, 38 or 39, respectively, preferably wherein the oligopeptide doesnot fully overlap with a PFAM domain if present, more preferably whereinthe oligopeptide does not overlap with a PFAM domain if present.

In a second aspect, provided is a method to synthesize a galactosylateddisaccharide or oligosaccharide by use of an N-acetylglucosamineb-1,X-galactosyltransferase as described herein.

In a preferred embodiment, the synthesis comprises the steps of:

-   -   a. providing UDP-galactose and any one of the        galactosyltransferase as defined herein, wherein the        galactosyltransferase is capable of transferring a galactose        residue from the UDP-galactose donor to one or more acceptor(s),        and    -   b. contacting any one of the galactosyltransferase and        UDP-galactose with one or more acceptor(s), under conditions        where the galactosyltransferase catalyzes the transfer of a        galactose residue from the UDP-galactose to the acceptor(s),    -   c. preferably, separating the galactosylated di- or        oligosaccharide.

In the scope of the disclosure, the wording “conditions where thegalactosyltransferase catalyzes the transfer of a galactose residuefrorn the UDP-galactose to the acceptor(s)” is to be understood to beconditions relating to physical or chemical parameters including but notlimited to temperature, pH, pressure, osmotic pressure andproduct/precursor/acceptor concentration.

In a particular embodiment, such conditions may include atemperature-range of 30+/−20 degrees centigrade, a pH-range of 7+/−3.

In a preferred embodiment of disclosure, the monosaccharideN-acetylglucosamine (GlcNAc), the monosaccharide N-acetylgalactosamine(GalNAc), a non-reducing N-acetylglucosamine of a di- and/oroligosaccharide (i.e., an N-acetylglucosamine at the non-reducing end ofa di- and/or oligosaccharide) and/or a non-reducingN-acetylgalactosamine of a di- and/or oligosaccharide (i.e., anN-acetylgalactosamine at the non-reducing end of a di- and/oroligosaccharide) are the acceptors of the N-acetylglucosamineb-1,X-galactosyltransferase. The di- and oligosaccharides having anon-reducing GlcNAc and/or GalNAc comprise di- and oligosaccharides,respectively, as defined herein.

Another preferred embodiment is a method as disclosed herein wherein anyone of the galactosyltransferase is contacted with at least twodifferent acceptors, preferably at least three different acceptors, morepreferably at least four different acceptors, even more preferably atleast five different acceptors, to synthesize a mixture ofgalactosylated disaccharides and/or oligosaccharides by use of aN-acetylglucosamine b-1,X-galactosyltransferase as described herein.According to the disclosure, the mixture comprises or consists of atleast two different “di- or oligosaccharides,” preferably at least threedifferent “di- or oligosaccharide,” more preferably at least fourdifferent “di- or oligosaccharide.” Preferably, the mixture comprises orconsists of neutral di-/oligosaccharides. More preferably, the mixturecomprises or consists of charged and/or neutral di- or oligosaccharides.In a preferred embodiment of the method and/or cell, the charged di- oroligosaccharides are sialylated di- or oligosaccharides. In a preferredembodiment of the method and/or cell, the neutral di- oroligosaccharides are fucosylated. In another preferred embodiment of themethod and/or cell, the neutral di- or oligosaccharides are notfucosylated.

The N-acetylglucosamine, N-acetylgalactosamine and/or di- and/oroligosaccharide containing an N-acetylglucosamine and/orN-acetylgalactosamine at the non-reducing end, i.e., the preferredacceptors of the N-acetylglucosamine b-1,X-galactosyltransferase(s) asdisclosed herein, are produced by methods comprising extraction fromnatural sources, biotechnological processes, physical processes,chemical processes, and combinations thereof.

In a further preferred embodiment of the method and/or cell, thegalactosylated disaccharide or oligosaccharide synthesized as describedherein is further glycosylated by providing a glycosyltransferase and anucleotide-sugar, which is donor for the glycosyltransferase. Theglycosyltransferase is preferably selected from the list comprising:fucosyltransferases, sialyltransferases, galactosyltransferases,glucosyltransferases, mannosyltransferases,N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases,N-acetylmannosaminyltransferases, xylosyltransferases,glucuronyltransferases, galacturonyltransferases,glucosaminyltransferases, N-glycolyineuraminyltransferases,rhamnnosyltransferases, N-acetylrhamnnosyltransferases,UDP-4-amnino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases,UDP-N-acetylglucosamine enolpyruvyl transferases andfucosaminyiltransferases.

In a more preferred embodiment of the method and/or cell, thefucosyltransferase is chosen from the list comprisingalpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase,alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase.

In another more preferred embodiment of the method and/or cell, thesialyltransferase is chosen from the list comprisingalpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase andalpha-2,8-sialyltransferase.

In another more preferred embodiment of the method and/or cell, thegalactosyltransferase is chosen from the list comprisingbeta-1,3-galactosyltransferase, N-acetylglucosaminebeta-1,3-galactosyltransferase, beta-1,4-galactosyltransferase,N-acetylglucosamine beta-1,4-galactosyltransferase,alpha-1,3-galactosyltransferase and alpha-1,4-galactosyltransferase.

In another more preferred embodiment of the method and/or cell, theglucosyltransferase is chosen from the list comprisingalpha-glucosyltransferase, beta-1,2-glucosyltransferase,beta-1,3-glucosyltransferase and beta-1,4-glucosyltransferase.

In another more preferred embodiment of the method and/or cell, themannosyltransferase is chosen from the list comprisingalpha-1,2-mannosyltransferase, alpha-1,3-mannosyltransferase andalpha-1,6-mannosyltransferase.

In another more preferred embodiment of the method and/or cell, theN-acetylglucosaminyltransferase is chosen from the list comprisinggalactoside beta-1,3-N-acetylglucosaminyltransferase andbeta-1,6-N-acetylglucosaminyltransferase.

In another more preferred embodiment of the method and/or cell, theN-acetylgalactosaminyltransferases is analpha-1,3-N-acetylgalactosaminyltransferase.

The nucleotide-sugar is preferably selected from the list comprisingGDP-fucose (GDP-Fuc), CMP-N-acetylneuraminic acid (CMP-Neu5Ac),UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-galactose (UDP-Gal),UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine(UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc),UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc orUDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,UDP-N-acetylfucosamine (UDP-L-FucNAc orUDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine(UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose),UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc orUDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose,CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N₃,CMP-Neu4,5Ac₂, CMP-Neu5,7Ac₂, CMP-Neu5,9Ac₂, CMP-Neu5,7(8,9)Ac₂,UDP-glucuronate, UDP-galacturonate, GDP-rhamnose and UDP-xylose.

In a preferred embodiment of the method and/or cell, the galactosylateddi- or oligosaccharide as described herein is additionally modified withone or more GleNAc moieties and the further glycosyltransferase is oneor more N-acetylglucosaminyltransferase(s), preferably a galactosidebeta-1,3-N-acetylglucosaminyltransferase and/or abeta-1,6-N-acetylglucosaminyltransferase and the nucleotide-activatedsugar is UDP-GlcNAc.

In an alternative, and/or additional preferred embodiment of the methodand/or cell, the galactosylated di- or oligosaccharide as describedherein is additionally sialylated, the further glycosyltransferase isone or more sialyltransferase(s), preferably analpha-2,3-sialyltransferase, an alpha-2,6-sialyltransferase and/or analpha-2,8-sialyltransferase, and the nucleotide-activated sugar isCMP-Neu5Ac and/or CMP-Neu5Gc.

In an alternative, and/or additional preferred embodiment of the methodand/or cell, the galactosylated di- or oligosaccharide as describedherein is additionally fucosylated, the further glycosyltransferase isone or more fucosyltransferase(s), preferably analpha-1,2-fucosyltransferase, an alpha-1,3-fucosyltransferase, analpha-1,4-fucosyltransferase and/or an alpha-1,6-fucosyltransferase, andthe nucleotide-activated sugar is GDP-fucose.

Preferably, at least two, more preferably at least three, even morepreferably at least four, even more preferably at least five, mostpreferably at least six, glycosyltransferases are provided to furtherglycosylate the galactosylated di- or oligosaccharide as synthesizedherein.

According to the disclosure, the galactosylated disaccharide oroligosaccharide (or further glycosylated form thereof as describedherein) can make use of a cell-free system, i.e., produced in acell-free system. Alternatively, the galactosylated disaccharide oroligosaccharide (or further glycosylated form thereof as describedherein) is produced by using a cell, preferably a single cell. Such cellcan be a non-metabolically engineered cell or a metabolically engineeredcell as disclosed herein.

In this context, it is a preferred embodiment that the cell is able toproduce (i) any one of the galactosyltransferase as defined herein,and/or (ii) UDP-galactose, and/or (iii) one or more acceptor(s) asdefined herein, wherein the cell is grown in a cultivation to obtain asufficient number of cells for use in the method according to thedisclosure at a desired scale. Upon growth of the cell to a desired celldensity, the cell is processed for use in the methods of the disclosure.For example, the cell is generally permeabilized or otherwise disruptedto allow entry of the saccharide acceptor(s) into the cell (if notproduced by the cell or if produced by another cell). Thegalactosyltransferase and/or UDP-galactose as produced by the cells can,however, in some situations, diffuse from the cells into theextracellular fluid or can be actively transported as described herein.Methods of permeabilizing cells so as to not significantly degradeenzymatic activity and nucleotide sugar stability are known to those ofskill in the art. A cell can be subjected to concentration, drying,lyophilization, treatment with surfactants, ultrasonic treatment,mechanical disruption, enzymatic treatment, and the like. The skilledperson will understand that one or more of (i) to (iii) can be producedby a same cell or by different cells. For example, one cell can providethe galactosyltransferase, while another cell can provide one or moreacceptors. Any one of (i) to (iii) can also be provided as such, i.e.,not produced by a cell. As such, the disclosure provides a method tosynthesize a galactosylated disaccharide or oligosaccharide, comprisingthe steps of a. providing UDP-galactose and any one of thegalactosyltransferase as defined herein, wherein thegalactosyltransferase is capable of transferring a galactose residuefrom the UDP-galactose donor to one or more acceptor(s), b. contactingany one of the galactosyltransferase and UDP-galactose with one or moreacceptor(s), under conditions where the galactosyltransferase catalyzesthe transfer of a galactose residue from the UDP-galactose to theacceptor(s), and c. preferably, separating the galactosylated di- oroligosaccharide; wherein at least one of the UDP-galactose,galactosyltransferase and acceptor(s) is provided, preferably produced,by a cell, preferably a single cell.

In this context, it is another preferred embodiment that thegalactosylated di- or oligosaccharide is produced by a cell, preferablya single cell. Such cell can be a non-metabolically engineered cell or ametabolically engineered cell as disclosed herein.

The N-acetylglucosamine, N-acetylgalactosamine and/or di- and/oroligosaccharide containing an N-acetylglucosamine and/orN-acetylgalactosamine at the non-reducing end being the acceptor(s) canbe taken up by the cell and/or synthesized by the cell. In the contextof the disclosure, it should be understood that the galactosylated di-or oligosaccharide according to the disclosure is preferably synthesizedintracellularly. The skilled person will further understand that afraction or substantially all of the synthesized galactosylated di- oroligosaccharide remains intracellularly and/or is excreted outside thecell either via passive or via active transport. As such, the disclosureprovides a method to synthesize a galactosylated disaccharide oroligosaccharide, comprising the steps of: a. providing a cell,preferably a single cell, as described herein (it is referred to thesecond and third aspect of disclosure), b. optionally providing one ormore acceptor(s) as described herein, c. cultivating and/or incubatingthe cell under conditions permissive for producing the galactosylateddi- or oligosaccharide, and d. preferably, separating the galactosylateddi- or oligosaccharide.

A third aspect of the disclosure relates to a cell that is metabolicallyengineered to synthesize a galactosylated disaccharide oroligosaccharide (or a further glycosylated form thereof as describedherein) by use of an N-acetylglucosamine b-1,X-galactosyltransferase asdisclosed herein. Throughout the application, unless explicitly statedotherwise, a “genetically modified cell” or “metabolically engineeredcell” preferably means a cell that is genetically modified ormetabolically engineered, respectively, for the production of agalactosylated di- or oligosaccharide according to the disclosure. Inthe context of the disclosure, the galactosylated di- or oligosaccharidepreferably does not occur in the wild type progenitor of themetabolically engineered cell.

Preferably, the cell as described herein (it is referred to the secondand third aspect of disclosure).

-   -   expresses any one of the N-acetylglucosamine        b-1,3-galactosyltransferases and/or N-acetylglucosamine        b-1,4-galactosyltransferases, and    -   is capable of synthesizing UDP-galactose (UDP-Gal) as donor for        the galactosyltransferases.

More preferably, the cell as described herein (it is referred to thesecond and third aspect of disclosure):

-   -   is capable of synthesizing one or more of the acceptor(s) as        disclosed herein, and    -   expresses any one of the N-acetylglucosamine        b-1,3-galactosyltransferases and/or N-acetylglucosamine        b-1,4-galactosyltransferases, and    -   is capable of synthesizing UDP-galactose (UDP-Gal) as donor for        the galactosyltransferases.

In another embodiment, the cell is capable of synthesizing one or morenucleotide-sugar donor(s) chosen from the list comprising: GDP-fucose(GDP-Fuc), CMP-N-acetylneuraminic acid (CMP-Neu5Ac),UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-galactose (UDP-Gal),UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine(UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc),UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc orUDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,UDP-N-acetylfucosamine (UDP-L-FucNAc orUDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine(UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose),UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc orUDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose,CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N₃,CMP-Neu4,5Ac₂, CMP-Neu5,7Ac₂, CMP-Neu5,9Ac₂, CMP-Neu5,7(8,9)Ac₂,UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose.

Alternatively or preferably, the cell is capable of expressing one ormore glycosyltransferases selected from the list comprising:fucosyltransferases, sialyltransferases, galactosyltransferases,glucosyltransferases, mannosyltransferases,N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases,N-acetylmannosaminyltransferases, xylosyltransferases,glucuronyltransferases, galacturonyltransferases,glucosaminyltransferases, N-glycolylneuraminyltransferases,rhamnosyltransferases, N-acetylrhamnosyltransferases,UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases,UDP-N-acetylglucosamine enolpyruvyl transferases andfucosaminyltransferases.

In a more preferred embodiment of the method and/or cell, the cell iscapable of expressing a fucosyltransferase chosen from the listcomprising alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase,alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase.

In another more preferred embodiment of the method and/or cell, the cellis capable of expressing a sialyltransferase chosen from the listcomprising alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase andalpha-2,8-sialyltransferase.

In another more preferred embodiment of the method and/or cell, the cellis capable of expressing a galactosyltransferase chosen from the listcomprising beta-1,3-galactosyltransferase, N-acetylglucosaminebeta-1,3-galactosyltransferase, beta-1,4-galactosyltransferase,N-acetylglucosamine beta-1,4-galactosyltransferase,alpha-1,3-galactosyltransferase and alpha-1,4-galactosyltransferase.

In another more preferred embodiment of the method and/or cell, the cellis capable of expressing a glucosyltransferase chosen from the listcomprising alpha-glucosyltransferase, beta-1,2-glucosyltransferase,beta-1,3-glucosyltransferase and beta-1,4-glucosyltransferase.

In another more preferred embodiment of the method and/or cell, the cellis capable of expressing a mannosyltransferase chosen from the listcomprising alpha-1,2-mannosyltransferase, alpha-1,3-mannosyltransferaseand alpha-1,6-manrnosyltransferase.

In another more preferred embodiment of the method and/or cell, the cellis capable of expressing an N-acetylglucosaminyltransferase chosen fromthe list comprising galactoside beta-1,3-N-acetylglucosaminyltransferaseand beta-1,6-N-acetylglucosaminyltransferase.

In another more preferred embodiment of the method and/or cell, the cellis capable of expressing a N-acetylgalactosaminyltransferase, which isan alpha-1,3-N-acetylgalactosaminyltransferase.

The glycosyltransferase family is a very broad family of enzymes capableof catalyzing the transfer of sugar moieties from activated donormolecules to specific acceptor molecules, forming glycosidic bonds. Aclassification of glycosyltransferases using nucleotide diphospho-sugar,nucleotide monophospho-sugar and sugar phosphates and related proteinsinto distinct sequence-based families has been described (Campbell etal., Biochem. J. 326, 929-939 (1997)) and is available on the CAZy(CArbohydrate-Active EnZymes) website (cazy.org).

Alternatively or preferably, the cell described herein is ametabolically engineered cell Preferably, the metabolically engineeredcell is modified with gene expression modules wherein the expressionfrom any one of the expression modules is constitutive or is created bya natural inducer.

The expression modules are also known as transcriptional units andcomprise polynucleotides for expression of recombinant genes includingcoding gene sequences and appropriate transcriptional and/ortranslational control signals that are operably linked to the codinggenes. The control signals comprise promoter sequences, untranslatedregions, ribosome binding sites, terminator sequences. The expressionmodules can contain elements for expression of one single recombinantgene but can also contain elements for expression of more recombinantgenes or can be organized in an operon structure for integratedexpression of two or more recombinant genes. The polynucleotides may beproduced by recombinant DNA technology using techniques well-known inthe art. Methods that are well known to those skilled in the art toconstruct expression modules include, for example, in vitro recombinantDNA techniques, synthetic techniques, and in vivo genetic recombination.See, for example, the techniques described in Sambrook et al. (2001)Molecular Cloning: a Laboratory Manual, 3rd Edition, Cold Spring HarborLaboratory Press, CSH, New York or to Current Protocols in MolecularBiology, John Wiley and Sons, N.Y. (1989 and yearly updates).

According to a preferred embodiment, the cell is modified with one ormore expression modules. The expression modules can be integrated in thegenome of the cell or can be presented to the cell on a vector. Thevector can be present in the form of a plasmid, cosmid, phage, liposome,or virus, which is to be stably transformed/transfected into themetabolically engineered cell Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. These vectors may contain selectionmarkers such as but not limited to antibiotic markers, auxotrophicmarkers, toxin-antitoxin markers, RNA sense/antisense markers. Theexpression system constructs may contain control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., see above. For recombinantproduction, cells can be genetically engineered to incorporateexpression systems or portions thereof or polynucleotides of thedisclosure. Introduction of a polynucleotide into the cell can beeffected by methods described in many standard laboratory manuals, suchas Davis et al., Basic Methods in Molecular Biology, (1986), andSambrook et al., 1989, supra.

As used herein an expression module comprises polynucleotides forexpression of at least one recombinant gene. The recombinant gene isinvolved in the expression of a polypeptide acting in the synthesis ofthe galactosylated di- or oligosaccharide like, e.g., aglycosyltransferase, a polypeptide directly involved in the synthesis ofa nucleotide-activated sugar or a membrane transporter protein asdescribed herein; or the recombinant gene is linked to other pathways inthe host cell that are not involved in the synthesis of thegalactosylated di- or oligosaccharide. The recombinant genes encodeendogenous proteins with a modified expression or activity, preferablythe endogenous proteins are overexpressed; or the recombinant genesencode heterologous proteins that are heterogeneously introduced andexpressed in the modified cell, preferably overexpressed. The endogenousproteins can have a modified expression in the cell that also expressesa heterologous protein.

According to a preferred embodiment of the disclosure, the expression ofeach of the expression modules is constitutive or created by a naturalinducer. As used herein, constitutive expression should be understood asexpression of a gene that is transcribed continuously in an organism.Expression that is created by a natural inducer should be understood asa facultative or regulatory expression of a gene that is only expressedupon a certain natural condition of the host (e.g., organism being inlabor, or during lactation), as a response to an environmental change(e.g., including but not limited to hormone, heat, cold, light,oxidative or osmotic stress/signaling), or dependent on the position ofthe developmental stage or the cell cycle of the host cell including butnot limited to apoptosis and autophagy.

According to a further embodiment, the recombinant polynucleotides areadapted to the codon usage of the respective cell or expression system.

In the method and cell described herein, the cell preferably comprisesmultiple copies of the same coding DNA sequence encoding for oneprotein. In the context of the disclosure, the protein can be aglycosyltransferase, a membrane transporter protein or any other proteinas disclosed herein. Throughout the application, the feature “multiple”means at least two, preferably at least three, more preferably at leastfour, even more preferably at least five.

In a preferred embodiment of the method and/or cell, the cell usedherein is genetically modified to produce a nucleotide-sugar selectedfrom the group: UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine(UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc),UDP-N-acetylmannosamine (UDP-ManNAc), GDP-fucose (GDP-Fuc), GDP-mannose(GDP-Man), UDP-glucose (UDP-Glc),UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc orUDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,UDP-N-acetylfucosamine (UDP-L-FucNAc orUDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine(UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose),UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc orUDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose,CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid(CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N₃, CMP-Neu4,5Ac₂, CMP-Neu5,7Ac₂,CMP-Neu5,9Ac₂, CMP-Neu5,7(8,9)Ac₂, UDP-glucuronate, UDP-galacturonate,GDP-rhamnose, or UDP-xylose. In a further embodiment, the cell isgenetically modified for the optimized production of any one of thenucleotide-sugars.

According to an embodiment of the method and/or cell, the cell iscapable of synthesizing or producing UDP-galactose. Preferably, the cellis optimized for UDP-galactose production. In an optional embodiment,the cell is modified in the expression or activity of the UDP-glucose4-epimerase GalE, which is capable of converting UDP-glucose intoUDP-galactose.

In a further embodiment, the nucleotide-sugar synthesized by the cell isUDP-galactose and the glycosyltransferase an N-acetylglucosamineb-1,3-galactosyltransferase or an N-acetylglucosamineb-1,4-galactosyltransferase.

In another preferred embodiment of the method and/or cell, the cell usedherein is capable of producing the nucleotide-sugar GDP-fucose. TheGDP-fucose can be provided by an enzyme expressed in the cell or by themetabolism of the cell. Such cell producing GDP-fucose can express anenzyme converting, e.g., fucose, which is to be added to the cell, toGDP-fucose. This enzyme may be, e.g., a bifunctional fucosekinase/fucose-1-phosphate guanylyltransferase, like Fkp from Bacteroidesfragilis, or the combination of one separate fucose kinase together withone separate fucose-1-phosphate guanylyltransferase like they are knownfrom several species including Homo sapiens, Sus scrofa and Rattusnorvegicus. Preferably, the cell is modified to produce GDP-fucose. Morepreferably, the cell is modified for enhanced GDP-fucose production. Themodification can be any one or more chosen from the group comprisingknock-out of a UDP-glucose:undecaprenyl-phosphate glucose-1-phosphatetransferase encoding gene, over-expression of a GDP-L-fucose synthaseencoding gene, over-expression of a GDP-mannose 4,6-dehydratase encodinggene, over-expression of a mannose-1-phosphate guanylyltransferaseencoding gene, over-expression of a phosphomannomutase encoding gene andover-expression of a mannose-6-phosphate isomerase encoding gene.

In another additive and/or alternative embodiment of the method and/orcell, the cell used herein is capable of producing the nucleotide-sugarCMP-N-acetylneuraminic acid (CMP-sialic acid). TheCMP-N-acetylneuraminic acid can be provided by an enzyme expressed inthe cell or by the metabolism of the cell. Preferably, the cell ismodified to produce CMP-N-acetylneuraminic acid. More preferably, thecell is modified for enhanced CMP-N-acetylneuraminic acid production.The modification can be one or more chosen from the group comprisingover-expression of a CMP-sialic acid synthetase encoding gene,over-expression of a sialate synthase encoding gene, and over-expressionof an N-acetyl-D-glucosamine 2-epimerase encoding gene.

Synthesis of CMP-N-acetylneuraminic acid makes use of GlcNAc but GlcNAcin the cell as described herein can be used as acceptor for theN-acetylglucosamine b-1,-X galactosyltransferases. Production ofCMP-N-acetylneuraminic acid in the cell may thus lower the GlcNAcavailable for the production of the saccharides of interest, i.e., agalactosylated di- or oligosaccharide. Production of bothCMP-N-acetylneuraminic acid and GlcNAc needs to be optimized to ensurehigh levels of both CMP-N-acetylneuraminic acid and GlcNAc. Suchoptimization may include efficient balancing and fine-tuning of theexpression levels of polypeptides involved in the synthesis of bothCMP-N-acetylneuraminic acid and GlcNAc.

In another additive or alternative embodiment of the method and/or cell,the cell as described herein is modified in the expression or activityof any glycosyltransferase expressed in the cell, preferably any of theglycosyltransferases as described herein.

In a preferred embodiment of the method and/or cell, the cell isgenetically modified in the expression or activity of an enzyme selectedfrom the group: a glucosamine 6-phosphate N-acetyltransferase, aphosphatase, a glycosyltransferase, anL-glutamine-D-fructose-6-phosphate aminotransferase, or aUDP-glucose-4-epimerase. According to the disclosure, the as suchenlisted enzymes comprising glucosamine 6-phosphate N-acetyltransferase,a phosphatase, a glycosyltransferase, anL-glutamine-D-fructose-6-phosphate aminotransferase, or a UDP-glucose4-epimerase are either endogenous proteins with a modified expression oractivity, preferably the endogenous proteins are overexpressed; or theenzymes of the as such enlisted group are heterologous proteins, whichcan be heterologously expressed by the cell. The heterologouslyexpressed proteins will then be introduced and expressed, preferablyoverexpressed. In another embodiment, the endogenous proteins can have amodified expression in the cell that also expresses a heterologousprotein. Heterologous expression can either be from the host's genome orfrom a vector introduced in the cell as described herein.

In another preferred embodiment, the cell described herein expresses atleast one glucosamine 6-phosphate N-acetyltransferase and a phosphataseto synthesize N-acetylglucosamine. In this preferred embodiment, theglucosamine 6-phosphate N-acetyltransferase is an enzyme capable ofconverting glucosamine-6-phosphate to N-acetylglucosamine-6-phosphate inthe cell and the phosphatase is capable of dephosphorylatingN-acetylglucosamine-6-phosphate to produce N-acetylglucosamine in thecell. In a more preferred embodiment, this phosphatase is a HAD-likephosphatase. Phosphatases from the HAD superfamily and the HAD-likefamily are described in the art. Examples from these families can befound in the enzymes expressed from genes yqaB, inhX, yniC, ybiV, yidA,ybjI, yigL or coffrom Escherichia coli or one or more of, e.g., the E.coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP,YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV,YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonasputida, ScDOG1 from S. cerevisiae or BsAraL from Bacillus subtilis asdescribed in WO18122225. One phosphatase that catalyzes this reaction isidentified in Blastocladiella emersonii. Phosphatases are generallynon-specific and the activity is generally not related to the family orstructure. Other examples can thus be found in all phosphatase families.Specific phosphatases are easily identified and screened by well-knownmethods as described by Fahs et a1. (ACS Chem. Biol. 11(11), 2944-2961(2016)). In a preferred embodiment, the phosphatase is encoded by aheterologous nucleic acid. In other words, the phosphatase is preferablyheterologously expressed in the cell. In another preferred embodiment,the glucosamine 6-phosphate N-acetyltransferase is encoded by aheterologous nucleic acid. In other words, the glucosamine 6-phosphateN-acetyltransferase is preferably heterologously expressed in the cell.

In a further preferred embodiment, the glucosamine 6-phosphateN-acetyltransferase is (i) a polypeptide sequence with UniProt IDP43577, or (ii) is a functional homologue, variant or derivative of thepolypeptide with UniProt ID P43577 having at least 80% overall sequenceidentity to the full-length of the polypeptide with UniProt ID P43577and having glucosamine 6-phosphate N-acetyltransferase activity, or(iii) is a functional fragment of the polypeptide with UniProt ID P43577and having glucosamine 6-phosphate N-acetyltransferase activity, or (iv)comprises a polypeptide comprising or consisting of an amino acidsequence having at least 80% sequence identity to the full-length aminoacid sequence of the polypeptide with UniProt ID P43577 and havingglucosamine 6-phosphate N-acetyltransferase activity. In anotherpreferred embodiment the L-glutamine-D-fructose-6-phosphateaminotransferase is (i) a polypeptide sequence with UniProt ID P17169,or (ii) is a functional homologue, variant or derivative of thepolypeptide with UniProt ID P17169 having at least 80% overall sequenceidentity to the full-length of the polypeptide with UniProt ID P17169and having L-glutamine D-fructose-6-phosphate aminotransferase activity,or (iii) is a functional fragment of the polypeptide with UniProt IDP17169 and having L-glutamine-D-fructose-6-phosphate aminotransferaseactivity, or (iv) comprises a polypeptide comprising or consisting of anamino acid sequence having at least 80% sequence identity to thefull-length amino acid sequence of the polypeptide with UniProt IDP17169 and having L-glutamine-D-fructose-6-phosphate aminotransferaseactivity. In an alternative preferred embodiment, theL-glutamine-D-fructose-6-phosphate aminotransferase (i) is a glmS*54polypeptide sequence differing from the wild-type E. coli glmS, havingUniProt ID P17169, by an A39T, an R250C and a G472S mutation asdescribed by Deng et a1. (Biochimie 88, 419-29 (2006) and havingL-glutamine-D-fructose-6-phosphate aminotransferase activity, or (ii) isa functional homologue, variant or derivative of the mutant glmS*54polypeptide differing from the wild-type E. coli glmS, having UniProt IDP17169, by an A39T, an R250C and a G472S mutation having at least 80%overall sequence identity to the full-length of the mutant glmS*54polypeptide differing from the wild-type E. coli glmS, having UniProt IDP17169, by an A39T, an R250C and a G472S mutation and havingL-glutamine-D-fructose-6-phosphate aminotransferase activity, or (iii)is a functional fragment of the mutant glmS*54 polypeptide differingfrom the wild-type E. coli glmS, having UniProt ID P17169, by an A39T,an R250C and a G472S mutation and havingL-glutamine-D-fructose-6-phosphate aminotransferase activity, or (iv)comprises a polypeptide comprising or consisting of an amino acidsequence having at least 80% sequence identity to the full-length aminoacid sequence of the mutant glmS*54 polypeptide differing from thewild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250Cand a G472S mutation and having L-glutamine-D-fructose-6-phosphateaminotransferase activity.

In another preferred embodiment of the method and/or cell, the cell isunable to convert N-acetylglucosamine-6-phosphate toglucosamine-6-phosphate, and/or unable to convertglucosamine-6-phosphate to fructose-6-phosphate. In a cellN-acetylglucosamine-6-phosphate can be converted toglucosamine-6-phosphate by activity of anN-acetylglucosamine-6-phosphate deacetylase like nagA andglucosamine-6-phosphate can be converted to fructose-6-phosphate byactivity of a glucosamine-6-phosphate deaminase like nagB. SuchN-acetylglucosamine-6-phosphate deacetylase and/orglucosamine-6-phosphate deaminase can obtain reduced expression orreduced activity or can be inactivated by mutagenesis or by partial orfull deletion of the corresponding polynucleotides encoding for thecoding sequences or by mutagenesis of the promoter sequences controllingthe expression of the corresponding encoding polynucleotides by methodswell-known in the art.

In a further preferred embodiment of the method and/or cell as describedherein, the cell is modified for enhanced UDP-galactose production andthe modification is chosen from the group comprising: knock-out of a5′-nucleotidase/UDP-sugar hydrolase encoding gene or knock-out of agalactose-1-phosphate uridylyltransferase encoding gene.

In a further preferred embodiment of the method and/or cell, the cell isusing a split metabolism having a production pathway and a biomasspathway as described in WO 2012/007481, which is herein incorporated byreference. The cell can, for example, be genetically modified toaccumulate fructose-6-phosphate by altering the genes selected from thephosphoglucoisomerase gene, phosphofructokinase gene,fructose-6-phosphate aldolase gene, fructose isomerase gene, and/orfructose:PEP phosphotransferase gene.

In a preferred embodiment of the method and/or cell according to thedisclosure, the cell expresses a membrane transporter protein or apolypeptide having transport activity hereby transporting compoundsacross the outer membrane of the cell wall. In another preferredembodiment of the method and/or cell, the cell expresses more than onemembrane transporter protein or polypeptide having transport activityhereby transporting compounds across the outer membrane of the cellwall. In a more preferred embodiment of the method and/or cell, the cellis modified in the expression or activity of the membrane transporterprotein or polypeptide having transport activity. The membranetransporter protein or polypeptide having transport activity is anendogenous protein of the cell with a modified expression or activity,preferably the endogenous membrane transporter protein or polypeptidehaving transport activity is overexpressed; alternatively the membranetransporter protein or polypeptide having transport activity is aheterologous protein that is heterogeneously introduced and expressed inthe cell, preferably overexpressed. The endogenous membrane transporterprotein or polypeptide having transport activity can have a modifiedexpression in the cell that also expresses a heterologous membranetransporter protein or polypeptide having transport activity.

In a more preferred embodiment of the method and/or cell, the membranetransporter protein or polypeptide having transport activity is chosenfrom the list comprising porters, P—P-bond-hydrolysis-driventransporters, β-barrel porins, auxiliary transport proteins, putativetransport proteins and phosphotransfer-driven group translocators. In aneven more preferred embodiment of the method and/or cell, the porterscomprise MFS transporters, sugar efflux transporters and siderophoreexporters. In another more preferred embodiment of the method and/orcell, the P—P-bond-hydrolysis-driven transporters comprise ABCtransporters and siderophore exporters.

In another preferred embodiment of the method and/or cell, the membranetransporter protein or polypeptide having transport activity controlsthe flow over the outer membrane of the cell wall of the galactosylateddisaccharide or oligosaccharide. In an alternative and or additionalpreferred embodiment of the method and/or cell, the membrane transporterprotein or polypeptide having transport activity controls the flow overthe outer membrane of the cell wall of a mixture of charged, preferablysialylated, and/or neutral di- and/or oligosaccharides comprising atleast one of the galactosylated disaccharide or oligosaccharide. In analternative and or additional preferred embodiment of the method and/orcell, the membrane transporter protein or polypeptide having transportactivity controls the flow over the outer membrane of the cell wall of amixture of charged, preferably sialylated, and/or neutraloligosaccharides comprising at least one of the galactosylatedoligosaccharide.

In an alternative and/or additional preferred embodiment of the methodand/or cell, the membrane transporter protein or polypeptide havingtransport activity controls the flow over the outer membrane of the cellwall of one or more precursor(s) to be used in the production of thegalactosylated disaccharide or oligosaccharide. In an alternative and/oradditional preferred embodiment of the method and/or cell, the membranetransporter protein or polypeptide having transport activity controlsthe flow over the outer membrane of the cell wall of one or moreprecursor(s) to be used in the production of the mixture of charged,preferably sialylated, and/or neutral di- and/or oligosaccharidescomprising at least one of the galactosylated disaccharide oroligosaccharide. In an alternative and/or additional preferredembodiment of the method and/or cell, the membrane transporter proteinor polypeptide having transport activity controls the flow over theouter membrane of the cell wall of one or more precursor(s) to be usedin the production of the mixture of charged, preferably sialylated,and/or neutral oligosaccharides comprising at least one of thegalactosylated oligosaccharide.

In an alternative and/or additional preferred embodiment of the methodand/or cell, the membrane transporter protein or polypeptide havingtransport activity controls the flow over the outer membrane of the cellwall of one or more acceptor(s) to be used in the production of thegalactosylated disaccharide or oligosaccharide. In an alternative and/oradditional preferred embodiment of the method and/or cell, the membranetransporter protein or polypeptide having transport activity controlsthe flow over the outer membrane of the cell wall of one or moreacceptor(s) to be used in the production of the mixture of charged,preferably sialylated, and/or neutral di- and/or oligosaccharidescomprising at least one of the galactosylated disaccharide oroligosaccharide. In an alternative and/or additional preferredembodiment of the method and/or cell, the membrane transporter proteinor polypeptide having transport activity controls the flow over theouter membrane of the cell wall of one or more acceptor(s) to be used inthe production of the mixture of charged, preferably sialylated, and/orneutral oligosaccharides comprising at least one of the galactosylatedoligosaccharide.

In another preferred embodiment of the method and/or cell, the membranetransporter protein or polypeptide having transport activity providesimproved production of the galactosylated disaccharide oroligosaccharide. In another preferred embodiment of the method and/orcell, the membrane transporter protein or polypeptide having transportactivity provides improved production of the mixture of charged,preferably sialylated, and/or neutral di- and/or oligosaccharidescomprising at least one of the galactosylated disaccharide oroligosaccharide. In another preferred embodiment of the method and/orcell, the membrane transporter protein or polypeptide having transportactivity provides improved production of the mixture of charged,preferably sialylated, and/or neutral oligosaccharides comprising atleast one of the galactosylated oligosaccharide.

In an alternative and/or additional preferred embodiment of the methodand/or cell, the membrane transporter protein or polypeptide havingtransport activity provides enabled efflux of the galactosylateddisaccharide or oligosaccharide. In an alternative and/or additionalpreferred embodiment of the method and/or cell, the membrane transporterprotein or polypeptide having transport activity provides enabled effluxof the mixture of charged, preferably sialylated, and/or neutral di-and/or oligosaccharides comprising at least one of the galactosylateddisaccharide or oligosaccharide. In an alternative and/or additionalpreferred embodiment of the method and/or cell, the membrane transporterprotein or polypeptide having transport activity provides enabled effluxof the mixture of charged, preferably sialylated, and/or neutraloligosaccharides comprising at least one of the galactosylatedoligosaccharide.

In an alternative and/or additional preferred embodiment of the methodand/or cell, the membrane transporter protein or polypeptide havingtransport activity provides enhanced efflux of the galactosylateddisaccharide or oligosaccharide. In an alternative and/or additionalpreferred embodiment of the method and/or cell, the membrane transporterprotein or polypeptide having transport activity provides enhancedefflux of the mixture of charged, preferably sialylated, and/or neutraldi- and/or oligosaccharides comprising at least one of thegalactosylated disaccharide or oligosaccharide. In an alternative and/oradditional preferred embodiment of the method and/or cell, the membranetransporter protein or polypeptide having transport activity providesenhanced efflux of the mixture of charged, preferably sialylated, and/orneutral oligosaccharides comprising at least one of the galactosylatedoligosaccharide.

In another preferred embodiment of the method and/or cell, the cellexpresses a polypeptide selected from the group comprising a lactosetransporter like, e.g., the LacY or lac12 permease, a glucosetransporter, a galactose transporter, a fucose transporter, atransporter for a nucleotide-activated sugar like, for example, atransporter for UDP-Gal, UDP-GlcNAc, GDP-Fuc or CMP-sialic acid. Assuch, the transporter internalizes a to the medium added precursorand/or acceptor for the synthesis of a galactosylated disaccharide oroligosaccharide, a mixture of charged, preferably sialylated, and/orneutral di- and/or oligosaccharides comprising at least one of thegalactosylated disaccharide or oligosaccharide or a mixture of charged,preferably sialylated, and/or neutral oligosaccharides comprising atleast one of the galactosylated oligosaccharide.

In a more preferred embodiment of the method and/or cell, the cellexpresses a membrane transporter protein belonging to the family of MFStransporters like, e.g., an MdfA polypeptide of the multidrugtransporter MdfA family from species comprising E. coli (UniProt IDPOAEY8), Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), Citrobacteryoungae (UniProt ID D4BC23) and Yokenella regensburgei (UniProt IDG9Z5F4). In another more preferred embodiment of the method and/or cell,the cell expresses a membrane transporter protein belonging to thefamily of sugar efflux transporters like, e.g., a SetA polypeptide ofthe SetA family from species comprising E. coli (UniProt ID P31675),Citrobacter koseri (UniProt ID AOA078LM16) and Klebsiella pneumoniae(UniProt ID A0A0C4MGS7). In another more preferred embodiment of themethod and/or cell, the cell expresses a membrane transporter proteinbelonging to the family of siderophore exporters like, e.g., the E. colientS (UniProt ID P24077) and the E. coli iceT (UniProt ID A0A024L207).In another more preferred embodiment of the method and/or cell, the cellexpresses a membrane transporter protein belonging to the family of ABCtransporters like, e.g., oppF from E. coli (UniProt ID P77737), lmrAfrom Lactococcus lactis subsp. lactis bv. diacetylactis (UniProt IDA0A1V0NEL4) and Blon_2475 from Bifidobacterium longum subsp. infantis(UniProt ID B7GPD4). In an even more preferred embodiment of the methodand/or cell, the cell expresses a membrane transporter protein chosenfrom the list comprising a lactose transporter like, e.g., the LacY orlac12 permease, a fucose transporter, a glucose transporter, a galactosetransporter, a transporter for a nucleotide-activated sugar like, forexample, a transporter for UDP-GlcNAc, UDP-Gal, GDP-Fuc and/orCMP-sialic acid.

In a preferred embodiment of the method and/or cell, the cell useslactose in a glycosylation reaction to produce an oligosaccharide.Lactose can be produced by the cell (for example, by the cell'smetabolism and/or upon metabolically engineering the cell for thispurpose as known to the skilled person), preferably intracellularly, orcan be added to the cell, which can import the lactose through passiveor active transport. Lactose production by a cell can be obtained byexpression of an N-acetylglucosamine beta-1,4-galactosyltransferase anda UDP-glucose 4-epimerase. More preferably, the cell is modified forenhanced lactose production. The modification can be any one or morechosen from the group comprising over-expression of anN-acetylglucosamine beta-1,4-galactosyltransferase, over-expression of aUDP-glucose 4-epimerase.

In a preferred embodiment of the method and/or cell of disclosure, acell using lactose as acceptor in a glycosylation reaction preferablyhas a transporter for the uptake of lactose from the cultivation. Morepreferably, the cell is optimized for lactose uptake. The optimizationcan be over-expression of a lactose transporter like a lactose permeasefrom E. coli or Kluyveromyces lactis. It is preferred the cellconstitutively expresses the lactose permease. Lactose can be added atthe start of the cultivation or it can be added as soon as enoughbiomass has been formed during the growth phase of the cultivation,i.e., the oligosaccharide production phase that is initiated by theaddition of lactose to the cultivation is decoupled from the growthphase. In a preferred embodiment, lactose is added at the start and/orduring the cultivation, i.e., the growth phase and production phase arenot decoupled.

In a preferred embodiment of the method and/or cell according to thedisclosure, the cell resists the phenomenon of lactose killing whengrown in an environment in which lactose is combined with one or moreother carbon source(s). With the term “lactose killing” is meant thehampered growth of the cell in medium in which lactose is presenttogether with another carbon source. In a preferred embodiment, the cellis genetically modified such that it retains at least 50% of the lactoseinflux without undergoing lactose killing, even at high lactoseconcentrations, as is described in WO 2016/075243. The geneticmodification comprises expression and/or over-expression of an exogenousand/or an endogenous lactose transporter gene by a heterologous promoterthat does not lead to a lactose killing phenotype and/or modification ofthe codon usage of the lactose transporter to create an alteredexpression of the lactose transporter that does not lead to a lactosekilling phenotype. The content of WO 2016/075243 in this regard isincorporated by reference. In the context of the disclosure, lactose ispreferably taken up by a cell as disclosed herein, wherein the lactoseis further glycosylated by a glycosyltransferase as disclosed herein tosynthesize a MMO, preferably a HMO.

According to another embodiment of the method and/or cell, the cell iscapable of producing phosphoenolpyruvate (PEP). According to anotherembodiment of the method and/or cell, the cell comprises a pathway forproduction of a galactosylated disaccharide or oligosaccharidecomprising a pathway for production of PEP. In a preferred embodiment ofthe method and/or cell, the cell is modified for enhanced productionand/or supply of PEP compared to a non-modified progenitor.

In another preferred embodiment, the cell comprises a pathway forproduction of a galactosylated disaccharide or oligosaccharidecomprising any one or more modifications for enhanced production and/orsupply of PEP compared to a non-modified progenitor.

In a preferred embodiment and as a means for enhanced production and/orsupply of PEP, one or more PEP-dependent, sugar-transportingphosphotransferase system(s) is/are disrupted, such as, but not limitedto: 1) the N-acetyl-D-glucosamine Npi-phosphotransferase (EC 2.7.1.193),which is, for instance, encoded by the nagE gene (or the clusternagABCD) in E. coli or Bacillus species, 2) ManXYZ, which encodes theEnzyme II Man complex (mannose PTS permease,protein-Npi-phosphohistidine-D-mannose phosphotransferase) that importsexogenous hexoses (mannose, glucose, glucosamine, fructose,2-deoxyglucose, mannosamine, N-acetylglucosamine, etc.) and releases thephosphate esters into the cell cytoplasm, 3) the glucose-specific PTStransporter (for instance, encoded by PtsG/Crr), which takes up glucoseand forms glucose-6-phosphate in the cytoplasm, 4) the sucrose-specificPTS transporter, which takes up sucrose and forms sucrose-6-phosphate inthe cytoplasm, 5) the fructose-specific PTS transporter (for instance,encoded by the genes fruA and fruB and the kinase fruK, which takes upfructose and forms in a first step fructose-1-phosphate and in a secondstep fructose-1,6 bisphosphate, 6) the lactose PTS transporter (forinstance, encoded by lacE in Lactococcus casei), which takes up lactoseand forms lactose-6-phosphate, 7) the galactitol-specific PTS enzyme,which takes up galactitol and/or sorbitol and formsgalactitol-1-phosphate or sorbitol-6-phosphate respectively, 8) themannitol-specific PTS enzyme, which takes up mannitol and/or sorbitoland forms mannitol-1-phosphate or sorbitol-6-phosphate respectively, and9) the trehalose-specific PTS enzyme, which takes up trehalose and formstrehalose-6-phosphate.

In another and/or additional preferred embodiment and as a means forenhanced production and/or supply of PEP, the full PTS system isdisrupted by disrupting the PtsIH/Crr gene cluster. PtsI (Enzyme I) is acytoplasmic protein that serves as the gateway for thephosphoenolpyruvate:sugar phosphotransferase system (PTSsugar) of E.coli K-12. PtsI is one of two (PtsI and PtsH) sugar non-specific proteinconstituents of the PTSsugar that, along with a sugar-specific innermembrane permease, effects a phosphotransfer cascade that results in thecoupled phosphorylation and transport of a variety of carbohydratesubstrates. HPr (histidine-containing protein) is one of twosugar-non-specific protein constituents of the PTSsugar. It accepts aphosphoryl group from phosphorylated Enzyme I (PtsI-P) and thentransfers it to the EIIA domain of any one of the many sugar-specificenzymes (collectively known as Enzymes II) of the PTSsugar. Crr orEIIAGIc is phosphorylated by PEP in a reaction requiring PtsH and PtsI.

In another and/or additional preferred embodiment, the cell is furthermodified to compensate for the deletion of a PTS system of a carbonsource by the introduction and/or overexpression of the correspondingpermease. These are, e.g., permeases or ABC transporters that comprisebut are not limited to transporters that specifically import lactosesuch as, e.g., the transporter encoded by the LacY gene from E. coli,sucrose such as, e.g., the transporter encoded by the cscB gene from E.coli, glucose such as, e.g., the transporter encoded by the galP genefrom E. coli, fructose such as, e.g., the transporter encoded by thefruI gene from Streptococcus mutans, or the Sorbitol/mannitol ABCtransporter such as the transporter encoded by the cluster SmoEFGK ofRhodobacter sphaeroides, the trehalose/sucrose/maltose transporter suchas the transporter encoded by the gene cluster ThuEFGK of Sinorhizobiummeliloti and the N-acetylglucosamine/galactose/glucose transporter suchas the transporter encoded by NagP of Shewanella oneidensis. Examples ofcombinations of PTS deletions with overexpression of alternativetransporters are: 1) the deletion of the glucose PTS system, e.g., ptsGgene, combined with the introduction and/or overexpression of a glucosepermease (e.g., galP of glcP), 2) the deletion of the fructose PTSsystem, e.g., one or more of the fruB, fruA, fruK genes, combined withthe introduction and/or overexpression of fructose permease, e.g., fruI,3) the deletion of the lactose PTS system, combined with theintroduction and/or overexpression of lactose permease, e.g., LacY,and/or 4) the deletion of the sucrose PTS system, combined with theintroduction and/or overexpression of a sucrose permease, e.g., cscB.

In a further preferred embodiment, the cell is modified to compensatefor the deletion of a PTS system of a carbon source by the introductionof carbohydrate kinases, such as glucokinase (EC 2.7.1.1, EC 2.7.1.2, EC2.7.1.63), galactokinase (EC 2.7.1.6), and/or fructokinase (EC 2.7.1.3,EC 2.7.1.4). Examples of combinations of PTS deletions withoverexpression of alternative transporters and a kinase are: 1) thedeletion of the glucose PTS system, e.g., ptsG gene, combined with theintroduction and/or overexpression of a glucose permease (e.g., galP ofglcP), combined with the introduction and/or overexpression of aglucokinase (e.g., glk), and/or 2) the deletion of the fructose PTSsystem, e.g., one or more of the fruB, fruA, fruK genes, combined withthe introduction and/or overexpression of fructose permease, e.g., fruI,combined with the introduction and/or overexpression of a fructokinase(e.g., frk or mak).

In another and/or additional preferred embodiment and as a means forenhanced production and/or supply of PEP, the cell is modified by theintroduction of or modification in any one or more of the listcomprising phosphoenolpyruvate synthase activity (EC: 2.7.9.2 encoded,for instance, in E. coli by ppsA), phosphoenolpyruvate carboxykinaseactivity (EC 4.1.1.32 or EC 4.1.1.49 encoded, for instance, inCorynebacterium glutamicum by PCK or in E. coli by pckA, resp.),phosphoenolpyruvate carboxylase activity (EC 4.1.1.31 encoded, forinstance, in E. coli by ppc), oxaloacetate decarboxylase activity (EC4.1.1.112 encoded, for instance, in E. coli by eda), pyruvate kinaseactivity (EC 2.7.1.40 encoded, for instance, in E. coli by pykA andpykF), pyruvate carboxylase activity (EC 6.4.1.1 encoded, for instance,in B. subtilis by pyc) and malate dehydrogenase activity (EC 1.1.1.38 orEC 1.1.1.40 encoded, for instance, in E. coli by maeA or maeB, resp.).

In a more preferred embodiment, the cell is modified to overexpress anyone or more of the polypeptides comprising ppsA from E. coli (UniProt IDP23538), PCK from C. glutamicum (UniProt ID Q6F5A5), pcka from E. coli(UniProt ID P22259), eda from E. coli (UniProt ID P0A955), maeA from E.coli (UniProt ID P26616), and maeB from E. coli (UniProt ID P76558).

In another and/or additional preferred embodiment, the cell is modifiedto express any one or more polypeptide having phosphoenolpyruvatesynthase activity, phosphoenolpyruvate carboxykinase activity,oxaloacetate decarboxylase activity, or malate dehydrogenase activity.

In another and/or additional preferred embodiment and as a means forenhanced production and/or supply of PEP, the cell is modified by areduced activity of phosphoenolpyruvate carboxylase activity, and/orpyruvate kinase activity, preferably a deletion of the genes encodingfor phosphoenolpyruvate carboxylase, the pyruvate carboxylase activityand/or pyruvate kinase.

In an exemplary embodiment, the cell is genetically modified bydifferent adaptations such as the overexpression of phosphoenolpyruvatesynthase combined with the deletion of a pyruvate kinase gene, theoverexpression of phosphoenolpyruvate synthase combined with thedeletion of a phosphoenolpyruvate carboxylase gene, the overexpressionof phosphoenolpyruvate synthase combined with the deletion of a pyruvatecarboxylase gene, the overexpression of phosphoenolpyruvatecarboxykinase combined with the deletion of a pyruvate kinase gene, theoverexpression of phosphoenolpyruvate carboxykinase combined with thedeletion of a phosphoenolpyruvate carboxylase gene, the overexpressionof phosphoenolpyruvate carboxykinase combined with the deletion of apyruvate carboxylase gene, the overexpression of oxaloacetatedecarboxylase combined with the deletion of a pyruvate kinase gene, theoverexpression of oxaloacetate decarboxylase combined with the deletionof a phosphoenolpyruvate carboxylase gene, the overexpression ofoxaloacetate decarboxylase combined with the deletion of a pyruvatecarboxylase gene, the overexpression of malate dehydrogenase combinedwith the deletion of a pyruvate kinase gene, the overexpression ofmalate dehydrogenase combined with the deletion of a phosphoenolpyruvatecarboxylase gene and/or the overexpression of malate dehydrogenasecombined with the deletion of a pyruvate carboxylase gene.

In another exemplary embodiment, the cell is genetically modified bydifferent adaptations such as the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase, the overexpression of phosphoenolpyruvate synthasecombined with the overexpression of an oxaloacetate decarboxylase, theoverexpression of phosphoenolpyruvate synthase combined with theoverexpression of a malate dehydrogenase, the overexpression ofphosphoenolpyruvate carboxykinase combined with the overexpression of anoxaloacetate decarboxylase, the overexpression of phosphoenolpyruvatecarboxykinase combined with the overexpression of a malatedehydrogenase, the overexpression of oxaloacetate decarboxylase combinedwith the overexpression of a malate dehydrogenase, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of aphosphoenolpyruvate carboxykinase and the overexpression of anoxaloacetate decarboxylase, the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase and the overexpression of a malate dehydrogenase, theoverexpression of phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase, the overexpression of phosphoenolpyruvatecarboxykinase combined with the overexpression of an oxaloacetatedecarboxylase and the overexpression of a malate dehydrogenase and/orthe overexpression of phosphoenolpyruvate synthase combined with theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase.

In another exemplary embodiment, the cell is genetically modified bydifferent adaptations such as the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase combined with the deletion of a pyruvate kinase gene, theoverexpression of phosphoenolpyruvate synthase combined with theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a pyruvate kinase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of amalate dehydrogenase combined with the deletion of a pyruvate kinasegene, the overexpression of phosphoenolpyruvate carboxykinase combinedwith the overexpression of an oxaloacetate decarboxylase combined withthe deletion of a pyruvate kinase gene, the overexpression ofphosphoenolpyruvate carboxykinase combined with the overexpression of amalate dehydrogenase combined with the deletion of a pyruvate kinasegene, the overexpression of an oxaloacetate decarboxylase combined withthe overexpression of a malate dehydrogenase combined with the deletionof a pyruvate kinase gene, the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase and the overexpression of an oxaloacetate decarboxylasecombined with the deletion of a pyruvate kinase gene, the overexpressionof phosphoenolpyruvate synthase combined with the overexpression of aphosphoenolpyruvate carboxykinase and the overexpression of a malatedehydrogenase combined with the deletion of a pyruvate kinase gene, theoverexpression of phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase combined with the deletion of a pyruvatekinase gene, the overexpression of a phosphoenolpyruvate carboxykinasecombined with the overexpression of an oxaloacetate decarboxylase andthe overexpression of a malate dehydrogenase combined with the deletionof a pyruvate kinase gene, the overexpression of phosphoenolpyruvatesynthase combined the overexpression of oxaloacetate decarboxylase andthe overexpression of a malate dehydrogenase combined with the deletionof a pyruvate kinase gene.

In another exemplary embodiment, the cell is genetically modified bydifferent adaptations such as the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase combined with the deletion of a phosphoenolpyruvatecarboxylase gene, the overexpression of phosphoenolpyruvate synthasecombined with the overexpression of an oxaloacetate decarboxylasecombined with the deletion of a phosphoenolpyruvate carboxylase gene,the overexpression of phosphoenolpyruvate synthase combined with theoverexpression of a malate dehydrogenase combined with the deletion of aphosphoenolpyruvate carboxylase gene, the overexpression of aphosphoenolpyruvate carboxykinase combined with the overexpression of anoxaloacetate decarboxylase combined with the deletion of aphosphoenolpyruvate carboxylase gene, the overexpression of aphosphoenolpyruvate carboxykinase combined with the overexpression of amalate dehydrogenase combined with the deletion of a phosphoenolpyruvatecarboxylase gene, the overexpression of an oxaloacetate decarboxylasecombined with the overexpression of a malate dehydrogenase combined withthe deletion of a phosphoenolpyruvate carboxylase gene, theoverexpression of phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a phosphoenolpyruvate carboxylase gene, the overexpressionof phosphoenolpyruvate synthase combined with the overexpression of aphosphoenolpyruvate carboxykinase and the overexpression of a malatedehydrogenase combined with the deletion of a phosphoenolpyruvatecarboxylase gene, the overexpression of phosphoenolpyruvate synthasecombined with the overexpression of a phosphoenolpyruvate carboxykinaseand the overexpression of an oxaloacetate decarboxylase and theoverexpression of a malate dehydrogenase combined with the deletion of aphosphoenolpyruvate carboxylase gene, the overexpression of aphosphoenolpyruvate carboxykinase combined with the overexpression of anoxaloacetate decarboxylase and the overexpression of a malatedehydrogenase combined with the deletion of a phosphoenolpyruvatecarboxylase gene, the overexpression of phosphoenolpyruvate synthasecombined the overexpression of an oxaloacetate decarboxylase and theoverexpression of a malate dehydrogenase combined with the deletion of aphosphoenolpyruvate carboxylase gene.

In another exemplary embodiment, the cell is genetically modified bydifferent adaptations such as the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase combined with the deletion of a pyruvate carboxylase gene,the overexpression of phosphoenolpyruvate synthase combined with theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a pyruvate carboxylase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of amalate dehydrogenase combined with the deletion of a pyruvatecarboxylase gene, the overexpression of a phosphoenolpyruvatecarboxykinase combined with the overexpression of an oxaloacetatedecarboxylase combined with the deletion of a pyruvate carboxylase gene,the overexpression of a phosphoenolpyruvate carboxykinase combined withthe overexpression of a malate dehydrogenase combined with the deletionof a pyruvate carboxylase gene, the overexpression of an oxaloacetatedecarboxylase combined with the overexpression of a malate dehydrogenasecombined with the deletion of a pyruvate carboxylase gene, theoverexpression of phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a pyruvate carboxylase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of aphosphoenolpyruvate carboxykinase and the overexpression of a malatedehydrogenase combined with the deletion of a pyruvate carboxylase gene,the overexpression of phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase combined with the deletion of a pyruvatecarboxylase gene, the overexpression of a phosphoenolpyruvatecarboxykinase combined with the overexpression of an oxaloacetatedecarboxylase and the overexpression of a malate dehydrogenase combinedwith the deletion of a pyruvate carboxylase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of anoxaloacetate decarboxylase and the overexpression of a malatedehydrogenase combined with the deletion of a pyruvate carboxylase gene.

In another exemplary embodiment, the cell is genetically modified bydifferent adaptations such as the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase combined with the deletion of a pyruvate kinase gene and aphosphoenolpyruvate carboxylase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of anoxaloacetate decarboxylase combined with the deletion of a pyruvatekinase gene and a phosphoenolpyruvate carboxylase gene, theoverexpression of phosphoenolpyruvate synthase combined with theoverexpression of a malate dehydrogenase combined with the deletion of apyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, theoverexpression of a phosphoenolpyruvate carboxykinase combined with theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylasegene, the overexpression of a phosphoenolpyruvate carboxykinase combinedwith the overexpression of a malate dehydrogenase combined with thedeletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylasegene, the overexpression of an oxaloacetate decarboxylase combined withthe overexpression of a malate dehydrogenase combined with the deletionof a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene,the overexpression of a phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylasegene, the overexpression of a phosphoenolpyruvate synthase combined withthe overexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of a malate dehydrogenase combined with the deletion of apyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, theoverexpression of a phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase combined with the deletion of a pyruvatekinase gene and a phosphoenolpyruvate carboxylase gene, theoverexpression of a phosphoenolpyruvate carboxykinase combined with theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase combined with the deletion of a pyruvatekinase gene and a phosphoenolpyruvate carboxylase gene, theoverexpression of a phosphoenolpyruvate synthase combined with theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase combined with the deletion of a pyruvatekinase gene and a phosphoenolpyruvate carboxylase gene.

In another exemplary embodiment, the cell is genetically modified bydifferent adaptations such as the overexpression of phosphoenolpyruvatesynthase combined with the overexpression of a phosphoenolpyruvatecarboxykinase combined with the deletion of a pyruvate kinase gene and apyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene,the overexpression of phosphoenolpyruvate synthase combined with theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a pyruvate kinase gene and a pyruvate carboxylase gene and aphosphoenolpyruvate carboxylase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of amalate dehydrogenase combined with the deletion of a pyruvate kinasegene and a pyruvate carboxylase gene and a phosphoenolpyruvatecarboxylase gene, the overexpression of a phosphoenolpyruvatecarboxykinase combined with the overexpression of an oxaloacetatedecarboxylase combined with the deletion of a pyruvate kinase gene and apyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene,the overexpression of a phosphoenolpyruvate carboxykinase combined withthe overexpression of a malate dehydrogenase combined with the deletionof a pyruvate kinase gene and a pyruvate carboxylase gene and aphosphoenolpyruvate carboxylase gene, the overexpression of anoxaloacetate decarboxylase combined with the overexpression of a malatedehydrogenase combined with the deletion of a pyruvate kinase gene and apyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene,the overexpression of phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase combined with thedeletion of a pyruvate kinase gene and a pyruvate carboxylase gene and aphosphoenolpyruvate carboxylase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of aphosphoenolpyruvate carboxykinase and the overexpression of a malatedehydrogenase combined with the deletion of a pyruvate kinase gene and apyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene,the overexpression of phosphoenolpyruvate synthase combined with theoverexpression of a phosphoenolpyruvate carboxykinase and theoverexpression of an oxaloacetate decarboxylase and the overexpressionof a malate dehydrogenase combined with the deletion of a pyruvatekinase gene and pyruvate carboxylase gene and a phosphoenolpyruvatecarboxylase gene, the overexpression of a phosphoenolpyruvatecarboxykinase combined with the overexpression of an oxaloacetatedecarboxylase and the overexpression of a malate dehydrogenase combinedwith the deletion of a pyruvate kinase gene and a pyruvate carboxylasegene and a phosphoenolpyruvate carboxylase gene, the overexpression ofphosphoenolpyruvate synthase combined with the overexpression of anoxaloacetate decarboxylase and the overexpression of a malatedehydrogenase combined with the deletion of a pyruvate kinase gene and apyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene.

According to another preferred embodiment of the method and/or cell, thecell comprises a modification for reduced production of acetate comparedto a non-modified progenitor. The modification can be any one or morechosen from the group comprising overexpression of an acetyl-coenzyme Asynthetase, a full or partial knock-out or rendered less functionalpyruvate dehydrogenase and a full or partial knock-out or rendered lessfunctional lactate dehydrogenase.

In a further embodiment of the method and/or cell, the cell is modifiedin the expression or activity of at least one acetyl-coenzyme Asynthetase like, e.g., acs from E. coli, S. cerevisiae, H. sapiens, M.musculus. In a preferred embodiment, the acetyl-coenzyme A synthetase isan endogenous protein of the cell with a modified expression oractivity, preferably the endogenous acetyl-coenzyme A synthetase isoverexpressed; alternatively, the acetyl-coenzyme A synthetase is aheterologous protein that is heterogeneously introduced and expressed inthe cell, preferably overexpressed. The endogenous acetyl-coenzyme Asynthetase can have a modified expression in the cell that alsoexpresses a heterologous acetyl-coenzyme A synthetase. In a morepreferred embodiment, the cell is modified in the expression or activityof the acetyl-coenzyme A synthetase acs from E. coli (UniProt IDP27550). In another and/or additional preferred embodiment, the cell ismodified in the expression or activity of a functional homolog, variantor derivative of acs from E. coli (UniProt ID P27550) having at least80% overall sequence identity to the full-length of the polypeptide fromE. coli (UniProt ID P27550) and having acetyl-coenzyme A synthetaseactivity.

In an alternative and/or additional further embodiment of the methodand/or cell, the cell is modified in the expression or activity of atleast one pyruvate dehydrogenase like, e.g., from E. coli, S.cerevisiae, H. sapiens and R. norvegicus. In a preferred embodiment, thecell has been modified to have at least one partially or fully knockedout or mutated pyruvate dehydrogenase encoding gene by means generallyknown by the person skilled in the art resulting in at least one proteinwith less functional or being disabled for pyruvate dehydrogenaseactivity. In a more preferred embodiment, the cell has a full knock-outin the poxB encoding gene resulting in a cell lacking pyruvatedehydrogenase activity.

In an alternative and/or additional further embodiment of the methodand/or cell, the cell is modified in the expression or activity of atleast one lactate dehydrogenase like, e.g., from E. coli, S. cerevisiae,H. sapiens and R. norvegicus. In a preferred embodiment, the cell hasbeen modified to have at least one partially or fully knocked out ormutated lactate dehydrogenase encoding gene by means generally known bythe person skilled in the art resulting in at least one protein withless functional or being disabled for lactate dehydrogenase activity. Ina more preferred embodiment, the cell has a full knock-out in the ldhAencoding gene resulting in a cell lacking lactate dehydrogenaseactivity.

According to another preferred embodiment of the method and/or cell, thecell comprises a lower or reduced expression and/or abolished, impaired,reduced or delayed activity of any one or more of the proteinscomprising beta-galactosidase, galactoside O-acetyltransferase,N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphatedeaminase, N-acetylglucosamine repressor, ribonucleotidemonophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphateglucose-1-phosphate transferase, L-fuculokinase, L-fucose isomerase,N-acetylneuraminate lyase, N-acetylmannosamine kinase,N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man,EIID-Man, ushA, galactose-1-phosphate uridylyltransferase,glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase,ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobicrespiration control protein, transcriptional repressor IclR, lonprotease, glucose-specific translocating phosphotransferase enzyme IIBCcomponent ptsG, glucose-specific translocating phosphotransferase (PTS)enzyme IIBC component malX, enzyme IIAGlc, beta-glucoside specific PTSenzyme II, fructose-specific PTS multiphosphoryl transfer protein FruAand FruB, ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formatelyase, acetate kinase, phosphoacyltransferase, phosphateacetyltransferase, pyruvate decarboxylase compared to a non-modifiedprogenitor.

According to another preferred embodiment of the method and/or cell, thecell comprises a catabolic pathway for selected mono-, di- oroligosaccharides, which is at least partially inactivated, the mono-,di-, or oligosaccharides being involved in and/or required for theproduction of a galactosylated disaccharide or oligosaccharide.

According to another preferred embodiment of the method and/or cell, thecell is using a precursor for the production of a galactosylateddisaccharide or oligosaccharide, preferably the precursor being fed tothe cell from the cultivation medium. According to a more preferredaspect of the method and/or cell, the cell is using at least twoprecursors for the production of the galactosylated disaccharide oroligosaccharide, preferably the precursors being fed to the cell fromthe cultivation medium. According to another preferred aspect of themethod and/or cell, the cell is producing at least one precursor,preferably at least two precursors, for the production of thegalactosylated disaccharide or oligosaccharide. In a preferredembodiment of the method and/or cell, the precursor that is used by thecell for the production of a galactosylated disaccharide oroligosaccharide is completely converted into the galactosylateddisaccharide or oligosaccharide.

According to another preferred embodiment of the method and/or cell, thecell produces 90 g/L or more of a galactosylated disaccharide oroligosaccharide in the whole broth and/or supernatant. In a morepreferred embodiment, the galactosylated disaccharide or oligosaccharideproduced in the whole broth and/or supernatant has a purity of at least80% measured on the total amount of the galactosylated disaccharide oroligosaccharide and its precursor produced by the cell in the wholebroth and/or supernatant, respectively.

In a preferred embodiment of the method and/or cell, the cell is capableof catabolizing a carbon source selected from the list comprising:glucose, fructose, mannose, galactose, lactose, sucrose, maltose,malto-oligosaccharides, trehalose, starch, cellulose, hemi-cellulose,corn-steep liquor, high-fructose syrup, glycerol, acetate, citrate,lactate, and pyruvate.

In an alternative embodiment of the method and/or cell, the cell asdescribed herein is capable of growing on a monosaccharide,disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, acomplex medium including molasses, corn steep liquor, peptone, tryptone,yeast extract or a mixture thereof like, e.g., a mixed feedstock,preferably a mixed monosaccharide feedstock like, e.g., hydrolyzedsucrose, as the main carbon source. With the term “complex medium” ismeant a medium for which the exact constitution is not determined. Withthe term main is meant the most important carbon source for thegalactosylated di- or oligosaccharide, biomass formation, carbon dioxideand/or by-products formation (e.g., acids and/or alcohols, such asacetate, lactate, and/or ethanol), i.e., 20, 30, 40, 50, 60, 70, 75, 80,85, 90, 95, 98, 99% of all the required carbon is derived from theabove-indicated carbon source. In one embodiment of the disclosure, thecarbon source is the sole carbon source for the organism, i.e., 100% ofall the required carbon is derived from the above-indicated carbonsource. Common main carbon sources comprise but are not limited toglucose, glycerol, fructose, maltose, lactose, arabinose,malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose,sucrose, galactose, mannose, methanol, ethanol, trehalose, starch,cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructosesyrup, acetate, citrate, lactate and pyruvate. With the term complexmedium is meant a medium for which the exact constitution is notdetermined. Examples are molasses, corn steep liquor, peptone, tryptoneor yeast extract.

Another embodiment of the disclosure provides for a method and a cellwherein a galactosylated di- or oligosaccharide is produced in and/or bya fungal, yeast, bacterial, insect, animal, plant or protozoan cell asdescribed herein. The cell is chosen from the list comprising abacterium, a yeast, a protozoan or a fungus, or, refers to a plant oranimal cell. The latter bacterium preferably belongs to the phylum ofthe Proteobacteria or the phylum of the Firmicutes or the phylum of theCyanobacteria or the phylum Deinococcus-Thermus. The latter bacteriumbelonging to the phylum Proteobacteria belongs preferably to the familyEnterobacteriaceae, preferably to the species Escherichia coli. Thelatter bacterium preferably relates to any strain belonging to thespecies Escherichia coli such as but not limited to Escherichia coli B,Escherichia coli C, Escherichia coli W, Escherichia coli K12,Escherichia coli Nissle. More specifically, the latter term relates tocultivated Escherichia coli strains—designated as E. coli K12strains—that are well-adapted to the laboratory environment, and, unlikewild type strains, have lost their ability to thrive in the intestine.Well-known examples of the E. coli K12 strains are K12 Wild type, W3110,MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200.Hence, the disclosure specifically relates to a mutated and/ortransformed Escherichia coli cell or strain as indicated above whereinthe E. coli strain is a K12 strain. More preferably, the Escherichiacoli K12 strain is E. coli MG1655. The latter bacterium belonging to thephylum Firmicutes belongs preferably to the Bacilli, preferablyLactobacilliales, with members such as Lactobacillus lactis, Leuconostocmesenteroides, or Bacillales with members such as from the genusBacillus, such as Bacillus subtilis or, B. amyloliquefaciens. The latterBacterium belonging to the phylum Actinobacteria, preferably belongingto the family of the Corynebacteriaceae, with members Corynebacteriumglutamicum or C. afermentans, or belonging to the family of theStreptomycetaceae with members Streptomyces griseus or S. fradiae. Thelatter yeast preferably belongs to the phylum of the Ascomycota or thephylum of the Basidiomycota or the phylum of the Deuteromycota or thephylum of the Zygomycetes. The latter yeast belongs preferably to thegenus Saccharomyces (with members like, e.g., Saccharomyces cerevisiae,S. bayanus, S. boulardii), Pichia (with members like, e.g., Pichiapastoris, P. anomala, P. kluyveri), Komagataella, Hansenula,Kluyveromyces (with members like, e.g., Kluyveromyces lactis, K.marxianus, K. thermotolerans), Debaromyces, Yarrowia (like, e.g.,Yarrowia lipolytica) or Starmerella (like, e.g., Starmerella bombicola).The latter yeast is preferably selected from Pichia pastoris, Yarrowialipolitica, Saccharomyces cerevisiae and Kluyveromyces lactis. Thelatter fungus belongs preferably to the genus Rhizopus, Dictyostelium,Penicillium, Mucor or Aspergillus. Plant cells include cells offlowering and non-flowering plants, as well as algal cells, for example,Chlamydomonas, Chlorella, etc. Preferably, the plant is a tobacco,alfalfa, rice, tomato, cotton, rapeseed, soy, maize or corn plant. Thelatter animal cell is preferably derived from non-human mammals (e.g.,cattle, buffalo, pig, sheep, mouse, rat), birds (e.g., chicken, duck,ostrich, turkey, pheasant), fish (e.g., swordfish, salmon, tuna, seabass, trout, catfish), invertebrates (e.g., lobster, crab, shrimp,clams, oyster, mussel, sea urchin), reptiles (e.g., snake, alligator,turtle), amphibians (e.g., frogs) or insects (e.g., fly, nematode) or isa genetically modified cell line derived from human cells excludingembryonic stem cells. Both human and non-human mammalian cells arepreferably chosen from the list comprising an epithelial cell like,e.g., a mammary epithelial cell, an embryonic kidney cell (e.g., HEK293or HEK 293T cell), a fibroblast cell, a COS cell, a Chinese hamsterovary (CHO) cell, a murine myeloma cell like, e.g., an N20, SP2/0 orYB2/0 cell, an NIH-3T3 cell, a non-mammary adult stem cell orderivatives thereof such as described in WO21067641. The latter insectcell is preferably derived from Spodoptera frugiperda like, e.g., Sf9 orSf21 cells, Bombyx mori, Mamestra brassicae, Trichoplusia ni like, e.g.,BTI-TN-5B1-4 cells or Drosophila melanogaster like, e.g., Drosophila S2cells. The latter protozoan cell preferably is a Leishmania tarentolaecell.

In a preferred embodiment of the method and/or cell, the cell is aviable Gram-negative bacterium that comprises a reduced or abolishedsynthesis of poly-N-acetyl-glucosamine (PNAG), Enterobacterial CommonAntigen (ECA), cellulose, colanic acid, core oligosaccharides,Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan,and/or trehalose compared to a non-modified progenitor.

In a more preferred embodiment of the method and/or cell, the reduced orabolished synthesis of poly-N-acetyl-glucosamine (PNAG), EnterobacterialCommon Antigen (ECA), cellulose, colanic acid, core oligosaccharides,Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan,and/or trehalose is provided by a mutation in any one or moreglycosyltransferases involved in the synthesis of any one of thepoly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA),cellulose, colanic acid, core oligosaccharides, OsmoregulatedPeriplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalose,wherein the mutation provides for a deletion or lower expression of anyone of the glycosyltransferases. The glycosyltransferases compriseglycosyltransferase genes encoding poly-N-acetyl-D-glucosamine synthasesubunits, UDP-N-acetylglucosamine-undecaprenyl-phosphateN-acetylglucosaminephosphotransferase, Fuc4NAc(4-acetamido-4,6-dideoxy-D-galactose) transferase,UDP-N-acetyl-D-mannosaminuronic acid transferase, theglycosyltransferase genes encoding the cellulose synthase catalyticsubunits, the cellulose biosynthesis protein, colanic acid biosynthesisglucuronosyltransferase, colanic acid biosynthesisgalactosyltransferase, colanic acid biosynthesis fucosyltransferase,UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase,putative colanic biosynthesis glycosyl transferase,UDP-glucuronate:LPS(HepIII) glycosyltransferase, ADP-heptose-LPSheptosyltransferase 2, ADP-heptose:LPS heptosyltransferase 1, putativeADP-heptose:LPS heptosyltransferase 4, lipopolysaccharide corebiosynthesis protein, UDP-glucose:(glucosyl)LPSα-1,2-glucosyltransferase, UDP-D-glucose:(glucosyl)LPSα-1,3-glucosyltransferase,UDP-D-galactose:(glucosyl)lipopolysaccharide-1,6-D-galactosyltransferase,lipopolysaccharide glucosyltransferase I, lipopolysaccharide coreheptosyltransferase 3, β-1,6-galactofuranosyltransferase,undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase,lipid IVA 4-amino-4-deoxy-L-arabinosyltransferase, bactoprenol glucosyltransferase, putative family 2 glycosyltransferase, the osmoregulatedperiplasmic glucans (OPG) biosynthesis protein G, OPG biosynthesisprotein H, glucosylglycerate phosphorylase, glycogen synthase,1,4-α-glucan branching enzyme, 4-α-glucanotransferase andtrehalose-6-phosphate synthase. In an exemplary embodiment, the cell ismutated in any one or more of the glycosyltransferases comprising pgaC,pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, wcaI, wcaJ,wcaL, waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ,wbbl, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsAand yaiP, wherein the mutation provides for a deletion or lowerexpression of any one of the glycosyltransferases.

In an alternative and/or additional preferred embodiment of the methodand/or cell, the reduced or abolished synthesis ofpoly-N-acetyl-glucosamine (PNAG) is provided by over-expression of acarbon storage regulator encoding gene, deletion of a Na+/H+ antiporterregulator encoding gene and/or deletion of the sensor histidine kinaseencoding gene.

According to another embodiment of the method of the disclosure, theconditions permissive to produce the galactosylated disaccharide oroligosaccharide comprise the use of a culture medium comprising at leastone precursor and/or acceptor for the production of the galactosylateddisaccharide or oligosaccharide. Preferably, the culture medium containsat least one precursor selected from the group comprising lactose,galactose, fucose, sialic acid, GlcNAc, GalNAc, lacto-N-biose (LNB),N-acetyllactosamine (LacNAc).

According to an alternative and/or additional embodiment of the methodof the disclosure, the conditions permissive to produce thegalactosylated disaccharide or oligosaccharide comprise adding to theculture medium at least one precursor and/or acceptor feed for theproduction of the galactosylated disaccharide or oligosaccharide.

According to an alternative embodiment of the method of the disclosure,the conditions permissive to produce the galactosylated disaccharide oroligosaccharide comprise the use of a culture medium to cultivate a cellof disclosure for the production of a galactosylated disaccharide oroligosaccharide wherein the culture medium lacks any precursor and/oracceptor for the production of the galactosylated disaccharide oroligosaccharide and is combined with a further addition to the culturemedium of at least one precursor and/or acceptor feed for the productionof the galactosylated disaccharide or oligosaccharide.

In a preferred embodiment, the method for the production of agalactosylated disaccharide or oligosaccharide as described hereincomprises at least one of the following steps:

-   -   i) Use of a culture medium comprising at least one precursor        and/or acceptor;    -   ii) Adding to the culture medium in a reactor at least one        precursor and/or acceptor feed wherein the total reactor volume        ranges from 250 mL (milliliter) to 10,000 m³ (cubic meter),        preferably in a continuous manner, and preferably so that the        final volume of the culture medium is not more than three-fold,        preferably not more than two-fold, more preferably less than        two-fold of the volume of the culture medium before the addition        of the precursor and/or acceptor feed;    -   iii) Adding to the culture medium in a reactor at least one        precursor and/or acceptor feed wherein the total reactor volume        ranges from 250 mL (milliliter) to 10,000 m³ (cubic meter),        preferably in a continuous manner, and preferably so that the        final volume of the culture medium is not more than three-fold,        preferably not more than two-fold, more preferably less than        two-fold of the volume of the culture medium before the addition        of the precursor and/or acceptor feed and wherein preferably,        the pH of the precursor and/or acceptor feed is set between 3        and 7 and wherein preferably, the temperature of the precursor        and/or acceptor feed is kept between 20° C. and 80° C.;    -   iv) Adding at least one precursor and/or acceptor feed in a        continuous manner to the culture medium over the course of 1        day, 2 days, 3 days, 4 days, 5 days by means of a feeding        solution;    -   v) Adding at least one precursor and/or acceptor feed in a        continuous manner to the culture medium over the course of 1        day, 2 days, 3 days, 4 days, 5 days by means of a feeding        solution and wherein preferably, the pH of the feeding solution        is set between 3 and 7 and wherein preferably, the temperature        of the feeding solution is kept between 20° C. and 80° C.;    -   the method resulting in a galactosylated disaccharide or        oligosaccharide with a concentration of at least 50 g/L,        preferably at least 75 g/L, more preferably at least 90 g/L,        more preferably at least 100 g/L, more preferably at least 125        g/L, more preferably at least 150 g/L, more preferably at least        175 g/L, more preferably at least 200 g/L in the final        cultivation broth.

In another and/or additional preferred embodiment, the method for theproduction of a galactosylated disaccharide or oligosaccharide asdescribed herein comprises at least one of the following steps:

-   -   i) Use of a culture medium comprising at least 50, more        preferably at least 75, more preferably at least 100, more        preferably at least 120, more preferably at least 150 gram of        lactose per liter of initial reactor volume wherein the reactor        volume ranges from 250 mL to 10,000 m³ (cubic meter);    -   ii) Adding to the culture medium at least one precursor and/or        acceptor in one pulse or in a discontinuous (pulsed) manner        wherein the total reactor volume ranges from 250 mL (milliliter)        to 10,000 m³ (cubic meter), preferably so that the final volume        of the culture medium is not more than three-fold, preferably        not more than two-fold, more preferably less than two-fold of        the volume of the culture medium before the addition of the        precursor and/or acceptor feed pulse(s);    -   iii) Adding to the culture medium in a reactor at least one        precursor and/or acceptor feed in one pulse or in a        discontinuous (pulsed) manner wherein the total reactor volume        ranges from 250 mL (milliliter) to 10,000 m³ (cubic meter),        preferably so that the final volume of the culture medium is not        more than three-fold, preferably not more than two-fold, more        preferably less than two-fold of the volume of the culture        medium before the addition of the precursor and/or acceptor feed        pulse(s) and wherein preferably, the pH of the precursor and/or        acceptor feed pulse(s) is set between 3 and 7 and wherein        preferably, the temperature of the precursor and/or acceptor        feed pulse(s) is kept between 20° C. and 80° C.;    -   iv) Adding at least one precursor and/or acceptor feed in a        discontinuous (pulsed) manner to the culture medium over the        course of 5 min., 10 min., 30 min., 1 hour, 2 hours, 4 hours, 10        hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means        of a feeding solution;    -   v) Adding at least one precursor and/or acceptor feed in a        discontinuous (pulsed) manner to the culture medium over the        course of 5 min., 10 min., 30 min., 1 hour, 2 hours, 4 hours, 10        hours, 12 hours, 1 day, 2 days, 3 days, 4 days, or 5 days by        means of a feeding solution and wherein preferably, the pH of        the feeding solution is set between 3 and 7 and wherein        preferably, the temperature of the feeding solution is kept        between 20° C. and 80° C.;    -   the method resulting in a galactosylated disaccharide or        oligosaccharide with a concentration of at least 50 g/L,        preferably at least 75 g/L, more preferably at least 90 g/L,        more preferably at least 100 g/L, more preferably at least 125        g/L, more preferably at least 150 g/L, more preferably at least        175 g/L, more preferably at least 200 g/L in the final        cultivation broth.

In a further embodiment of the methods described herein the host cellsare cultivated for at least about 60, 80, 100, or about 120 hours or ina continuous manner.

In a preferred embodiment, a carbon source is provided, preferablysucrose, in the culture medium for 3 or more days, preferably up to 7days; and/or provided, in the culture medium, at least 100,advantageously at least 105, more advantageously at least 110, even moreadvantageously at least 120 grams of sucrose per liter of initialculture volume in a continuous manner, so that the final volume of theculture medium is not more than three-fold, advantageously not more thantwo-fold, more advantageously less than two-fold of the volume of theculturing medium before the culturing.

Preferably, when performing the method as described herein, a firstphase of exponential cell growth is provided by adding a carbon source,preferably glucose or sucrose, to the culture medium before theprecursor is added to the cultivation in a second phase.

In another preferred embodiment of the method of disclosure, a firstphase of exponential cell growth is provided by adding a carbon-basedsubstrate, preferably glucose or sucrose, to the culture mediumcomprising a precursor, followed by a second phase wherein only acarbon-based substrate, preferably glucose or sucrose, is added to thecultivation.

In another preferred embodiment of the method of disclosure, a firstphase of exponential cell growth is provided by adding a carbon-basedsubstrate, preferably glucose or sucrose, to the culture mediumcomprising a precursor, followed by a second phase wherein acarbon-based substrate, preferably glucose or sucrose, and a precursorare added to the cultivation.

In an alternative preferable embodiment, in the method as describedherein, the precursor is added already in the first phase of exponentialgrowth together with the carbon-based substrate.

According to the disclosure, the method as described herein preferablycomprises a step of separating the galactosylated di- oroligosaccharide. The term “separating” means harvesting, collecting, orretrieving the galactosylated di- or oligosaccharide from the enzymereaction or the cell and/or the medium of its growth.

The galactosylated di- or oligosaccharide can be separated in aconventional manner from the enzyme mixture or the aqueous culturemedium, in which the cell was grown. In case the saccharide is stillpresent in the cells producing the saccharide, conventional manners tofree or to extract the saccharide out of the cells can be used, such ascell destruction using high pH, heat shock, sonication, French press,homogenization, enzymatic hydrolysis, chemical hydrolysis, solventhydrolysis, detergent, hydrolysis, etc. The enzyme reaction mixture, theculture medium and/or cell extract together and separately can then befurther used for separating the saccharide. This preferably involvesclarifying the saccharide-containing mixture to remove suspendedparticulates and contaminants, particularly cells, cell components,insoluble metabolites and debris produced by culturing the geneticallymodified cell. In this step, the saccharide-containing mixture can beclarified in a conventional manner. Preferably, thesaccharide-containing mixture is clarified by centrifugation,flocculation, decantation and/or filtration. Another step of separatingthe saccharide from the saccharide-containing mixture preferablyinvolves removing substantially all the proteins, as well as peptides,amino acids, RNA and DNA and any endotoxins and glycolipids that couldinterfere with the subsequent separation step, from thesaccharide-containing mixture, preferably after it has been clarified.In this step, proteins and related impurities can be removed from thesaccharide-containing mixture in a conventional manner. Preferably,proteins, salts, by-products, color, endotoxins and other relatedimpurities are removed from the saccharide-containing mixture byultrafiltration, nanofiltration, two-phase partitioning, reverseosmosis, microfiltration, activated charcoal or carbon treatment,treatment with non-ionic surfactants, enzymatic digestion, tangentialflow high-performance filtration, tangential flow ultrafiltration,electrophoresis (e.g., using slab-polyacrylamide or sodium dodecylsulphate-polyacrylamide gel electrophoresis (PAGE)), affinitychromatography (using affinity ligands including, e.g., DEAE-Sepharose,poly-L-lysine and polymyxin-B, endotoxin-selective adsorber matrices),ion exchange chromatography (e.g., but not limited to cation exchange,anion exchange, mixed bed ion exchange, inside-out ligand attachment),hydrophobic interaction chromatography and/or gel filtration (i.e., sizeexclusion chromatography), particularly by chromatography, moreparticularly by ion exchange chromatography or hydrophobic interactionchromatography or ligand exchange chromatography. With the exception ofsize exclusion chromatography, proteins and related impurities areretained by a chromatography medium or a selected membrane, thesaccharide remains in the saccharide-containing mixture.

In a further preferred embodiment, the methods as described herein alsoprovide for a further purification of the galactosylated di- oroligosaccharide. A further purification of the saccharide may beaccomplished, for example, by use of (activated) charcoal or carbon,nanofiltration, ultrafiltration or ion exchange to remove any remainingDNA, protein, LPS, endotoxins, or other impurity. Alcohols, such asethanol, and aqueous alcohol mixtures can also be used. Anotherpurification step is accomplished by crystallization, evaporation orprecipitation of the product. Another purification step is to dry, e.g.,spray dry, lyophilize, spray freeze dry, freeze spray dry, band dry,belt dry, vacuum band dry, vacuum belt dry, drum dry, roller dry, vacuumdrum dry or vacuum roller dry the produced saccharide.

In an exemplary embodiment, the separation and purification is made in aprocess, comprising the following steps in any order:

-   -   a) contacting the cultivation or a clarified version thereof        with a nanofiltration membrane with a molecular weight cut-off        (MWCO) of 600-3500 Da ensuring the retention of the produced        saccharide and allowing at least a part of the proteins, salts,        by-products, color and other related impurities to pass,    -   b) conducting a diafiltration process on the retentate from step        a), using the membrane, with an aqueous solution of an inorganic        electrolyte, followed by optional diafiltration with pure water        to remove excess of the electrolyte, and    -   c) collecting the retentate enriched in the saccharide in the        form of a salt from the cation of the electrolyte and preferably        spray drying the retentate.

In an alternative exemplary embodiment, the separation and purificationof the produced galactosylated di- or oligosaccharide is made in aprocess, comprising the following steps in any order: subjecting thecultivation or a clarified version thereof to two membrane filtrationsteps using different membranes, wherein, one membrane has a molecularweight cut-off of between about 300 to about 500 Dalton, and the othermembrane has a molecular weight cut-off of between about 600 to about800 Dalton, and preferably spray drying.

In an alternative exemplary embodiment, the separation and purificationof the produced galactosylated di- or oligosaccharide is made in aprocess, comprising the following steps in any order comprising the stepof treating the cultivation or a clarified version thereof with a strongcation exchange resin in H+−form and a weak anion exchange resin in freebase form, and preferably spray drying.

In an alternative exemplary embodiment, the separation and purificationof the produced galactosylated di- or oligosaccharide is made in thefollowing way. The cultivation comprising the produced oligosaccharide,biomass, medium components and contaminants is applied to the followingpurification steps:

-   -   i) separation of biomass from the cultivation,    -   ii) cationic ion exchanger treatment for the removal of        positively charged material,    -   iii) anionic ion exchanger treatment for the removal of        negatively charged material, and    -   iv) nanofiltration step and/or electrodialysis step,    -   wherein a purified solution comprising the produced saccharide        at a purity of greater than or equal to 80 percent is provided.        Optionally the purified solution is dried by any one or more        drying steps chosen from the list comprising spray drying,        lyophilization, spray freeze drying, freeze spray drying, band        drying, belt drying, vacuum band drying, vacuum belt drying,        drum drying, roller drying, vacuum drum drying and vacuum roller        drying.

In an alternative exemplary embodiment, the separation and purificationof the produced galactosylated di- or oligosaccharide is made in aprocess, comprising the following steps in any order: enzymatictreatment of the cultivation; removal of the biomass from thecultivation; ultrafiltration; nanofiltration; and a columnchromatography step. Preferably, such column chromatography is a singlecolumn or a multiple column. Further preferably, the columnchromatography step is simulated moving bed chromatography. Suchsimulated moving bed chromatography preferably comprises i) at least 4columns, wherein at least one column comprises a weak or strong cationexchange resin; and/or ii) four zones I, II, III and IV with differentflow rates; and/or iii) an eluent comprising water; and/or iv) anoperating temperature of 15 degrees to 60 degrees centigrade. Optionallythe process further comprises a step of drying chosen from the listcomprising spray drying, lyophilization, spray freeze drying, freezespray drying, band drying, belt drying, vacuum band drying, vacuum beltdrying, drum drying, roller drying, vacuum drum drying and vacuum rollerdrying.

In a specific embodiment, the disclosure provides the producedgalactosylated di- or oligosaccharide, which is dried to powder by anyone or more drying steps chosen from the list comprising spray drying,lyophilization, spray freeze drying, freeze spray drying, band drying,belt drying, vacuum band drying, vacuum belt drying, drum drying, rollerdrying, vacuum drum drying and vacuum roller drying, wherein the driedpowder contains <15 percent-wt. of water, preferably <10 percent-wt. ofwater, more preferably <7 percent-wt. of water, most preferably <5percent-wt. of water.

For identification of the produced galactosylated di- or oligosaccharideas described herein, the monomeric building blocks (e.g., themonosaccharide or glycan unit composition), the anomeric configurationof side chains, the presence and location of substituent groups, degreeof polymerization/molecular weight and the linkage pattern can beidentified by standard methods known in the art, such as, e.g.,methylation analysis, reductive cleavage, hydrolysis, GC-MS (gaschromatography-mass spectrometry), MALDI-MS (Matrix-assisted laserdesorption/ionization-mass spectrometry), ESI-MS (Electrosprayionization-mass spectrometry), HPLC (High-Performance Liquidchromatography with ultraviolet or refractive index detection),HPAEC-PAD (High-Performance Anion-Exchange chromatography with PulsedAmperometric Detection), CE (capillary electrophoresis), IR(infrared)/Raman spectroscopy, and NMR (Nuclear magnetic resonance)spectroscopy techniques. The crystal structure can be solved using,e.g., solid-state NMR, FT-IR (Fourier transform infrared spectroscopy),and WAXS (wide-angle X-ray scattering). The degree of polymerization(DP), the DP distribution, and polydispersity can be determined by,e.g., viscosimetry and SEC (SEC-HPLC, high performance size-exclusionchromatography). To identify the monomeric components of the saccharidemethods such as, e.g., acid-catalyzed hydrolysis, HPLC (high performanceliquid chromatography) or GLC (gas-liquid chromatography) (afterconversion to alditol acetates) may be used. To determine the glycosidiclinkages, the saccharide is methylated with methyl iodide and strongbase in DMSO, hydrolysis is performed, a reduction to partiallymethylated alditols is achieved, an acetylation to methylated alditolacetates is performed, and the analysis is carried out by GLC/MS(gas-liquid chromatography coupled with mass spectrometry). To determinethe saccharide sequence, a partial depolymerization is carried out usingan acid or enzymes to determine the structures. To identify the anomericconfiguration, the saccharide is subjected to enzymatic analysis, e.g.,it is contacted with an enzyme that is specific for a particular type oflinkage, e.g., beta-galactosidase, or alpha-glucosidase, etc., and NMRmay be used to analyze the products.

Provided is the use of the N-acetylglucosamineb-1,X-galactosyltransferases as described herein for the synthesis of agalactosylated disaccharide or oligosaccharide. In a preferredembodiment, described is use of the N-acetylglucosamineb-1,X-galactosyltransferases as described herein for the synthesis of amixture of charged, preferably sialylated, and/or neutral di- and/oroligosaccharides comprising at least one galactosylated disaccharide oroligosaccharide. In another preferred embodiment, the disclosureprovides the use of the N-acetylglucosamine b-1,X-galactosyltransferasesas described herein for the synthesis of a mixture of charged,preferably sialylated, and/or neutral oligosaccharides comprising atleast one galactosylated oligosaccharide.

Also provided for is the use of a metabolically engineered cell asdescribed herein for the production of a galactosylated disaccharide oroligosaccharide. In a preferred embodiment, the disclosure provides theuse of a metabolically engineered cell as described herein for theproduction of a mixture of charged, preferably sialylated, and/orneutral di- and/or oligosaccharides comprising at least onegalactosylated disaccharide or oligosaccharide. In another preferredembodiment, the disclosure also provides for the use of a metabolicallyengineered cell as described herein for the production of a mixture ofcharged, preferably sialylated, and/or neutral oligosaccharidescomprising at least one galactosylated oligosaccharide. In anotherpreferred embodiment, a metabolically engineered cell as describedherein is used for the production of a galactosylated disaccharide oroligosaccharide. In another preferred embodiment, a metabolicallyengineered cell as described herein is used for the production of amixture of charged, preferably sialylated, and/or neutral di- and/oroligosaccharides comprising at least one galactosylated disaccharide oroligosaccharide. In another preferred embodiment, a metabolicallyengineered cell as described herein is used for the production of amixture of charged, preferably sialylated, and/or neutraloligosaccharides comprising at least one galactosylated oligosaccharide.

Also provided for is the use of a method as described herein forproducing a galactosylated disaccharide or oligosaccharide. In apreferred embodiment, the disclosure provides the use of a method asdescribed herein for the production of a mixture of charged, preferablysialylated, and/or neutral di- and/or oligosaccharides comprising atleast one galactosylated disaccharide or oligosaccharide. In anotherpreferred embodiment, the disclosure also provides for the use of amethod as described herein for the production of a mixture of charged,preferably sialylated, and/or neutral oligosaccharides comprising atleast one galactosylated oligosaccharide.

Products comprising the galactosylated oligosaccharides (or furtherglycosylated form thereof) produced in the disclosure

In some embodiments, the galactosylated oligosaccharide (or furtherglycosylated form thereof) produced as described herein is incorporatedinto a food (e.g., human food or feed), dietary supplement,pharmaceutical ingredient, cosmetic ingredient or medicine. In someembodiments, the galactosylated oligosaccharide (or further glycosylatedform thereof) is mixed with one or more ingredients suitable for food,feed, dietary supplement, pharmaceutical ingredient, cosmetic ingredientor medicine.

In some embodiments, the dietary supplement comprises at least oneprebiotic ingredient and/or at least one probiotic ingredient.

A “prebiotic” is a substance that promotes growth of microorganismsbeneficial to the host, particularly microorganisms in thegastrointestinal tract. In some embodiments, a dietary supplementprovides multiple prebiotics, including the galactosylatedoligosaccharide produced and/or purified by a process disclosed in thisspecification, to promote growth of one or more beneficialmicroorganisms. Examples of prebiotic ingredients for dietarysupplements include other prebiotic molecules (e.g., HMOs) and plantpolysaccharides (e.g., inulin, pectin, b-glucan andxylooligosaccharide). A “probiotic” product typically contains livemicroorganisms that replace or add to gastrointestinal microflora, tothe benefit of the recipient. Examples of such microorganisms includeLactobacillus species (for example, L. acidophilus and L. bulgaricus),Bifidobacterium species (for example, B. animalis, B. longum and B.infantis (e.g., Bi-26)), and Saccharomyces boulardii. In someembodiments, a galactosylated oligosaccharide produced and/or purifiedby a process of this specification is orally administered in combinationwith such microorganism.

Examples of further ingredients for dietary supplements includedisaccharides (e.g., lactose), monosaccharides (e.g., glucose andgalactose), thickeners (e.g., gum Arabic), acidity regulators (e.g.,trisodium citrate), water, skimmed milk, and flavorings.

In some embodiments, the galactosylated oligosaccharide (or furtherglycosylated form thereof) is incorporated into a human baby food (e.g.,infant formula). Infant formula is generally a manufactured food forfeeding to infants as a complete or partial substitute for human breastmilk. In some embodiments, infant formula is sold as a powder andprepared for bottle- or cup-feeding to an infant by mixing with water.The composition of infant formula is typically designed to roughly mimichuman breast milk. In some embodiments, a galactosylated oligosaccharide(or further glycosylated form thereof) produced and/or purified by aprocess herein is included in infant formula to provide nutritionalbenefits similar to those provided by the oligosaccharides in humanbreast milk. In some embodiments, the galactosylated oligosaccharide ismixed with one or more ingredients of the infant formula. Examples ofinfant formula ingredients include nonfat milk, carbohydrate sources(e.g., lactose), protein sources (e.g., whey protein concentrate andcasein), fat sources (e.g., vegetable oils—such as palm, high oleicsafflower oil, rapeseed, coconut and/or sunflower oil; and fish oils),vitamins (e.g., vitamins A, Bb, Bi2, C and D), minerals (e.g., potassiumcitrate, calcium citrate, magnesium chloride, sodium chloride, sodiumcitrate and calcium phosphate) and possibly human milk oligosaccharides(HMOs). Such HMOs may include, for example, DiFL, lacto-N-triose II,LNT, LNnT, lacto-N-fucopentaose I, lacto-N-neofucopentaose,lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaoseV, lacto-N-neofucopentaose V, lacto-N-difucohexaose I,lacto-N-difucohexaose II, 6′-galactosyllactose, 3′-galactosyllactose,lacto-N-hexaose and lacto-N-neohexaose.

In some embodiments, the one or more infant formula ingredients comprisenon-fat milk, a carbohydrate source, a protein source, a fat source,and/or a vitamin and mineral.

In some embodiments, the one or more infant formula ingredients compriselactose, whey protein concentrate and/or high oleic safflower oil.

In some embodiments, the concentration of the galactosylatedoligosaccharide (or further glycosylated form thereof) in the infantformula is approximately the same concentration as the oligosaccharide'sconcentration generally present in human breast milk. In someembodiments, the concentration of the galactosylated oligosaccharide inthe infant formula is approximately the same concentration as theconcentration of that oligosaccharide generally present in human breastmilk.

In some embodiments, the galactosylated oligosaccharide (or furtherglycosylated form thereof) is incorporated into a feed preparation,wherein the feed is chosen from the list comprising pet food, animalmilk replacer, veterinary product, post weaning feed, and creep feed.

In some embodiments, a food, a feed, a dietary supplement, apharmaceutical ingredient and/or a medicine comprises at least oneimmunomodulatory ingredient.

“An immunomodulatory” ingredient is a substance that modifies the immuneresponse or the functioning of the immune system. An “immunomodulatory”ingredient can modify the immune response by an increase(immunostimulators) or a decrease (immunosuppressives) of the productionof serum antibodies. Immunostimulators are used, among others, toenhance the immune response against infectious diseases, tumors, primaryor secondary immunodeficiency, and alterations in antibody transfer.Immunosuppressive ingredients are used to reduce the immune responseagainst transplanted organs and to treat autoimmune diseases such aspemphigus, lupus, or allergies. In some embodiments, theimmunomodulatory ingredient has anti-inflammatory activity. In someembodiments, a food, a feed, a dietary supplement, a pharmaceuticalingredient and/or a medicine provides multiple immunomodulators,including the galactosylated oligosaccharide (or further glycosylatedform thereof) produced and/or purified by a process disclosed in thisspecification, to adapt the immune system for proper functioning.Immunity varies strongly in distinguishable life stages. Different foodcomponents can affect specific immune reactions, depending on thecharacteristics of deviating metabolic processes, and consumers andpatients. A food supplemented with an immunomodulatory ingredient isalso called a functional food. A functional food is a food product withspecific health benefits for specific groups of consumers. Examples ofimmunomodulatory ingredients present in functional food as well as infeed and dietary supplements comprise other immunomodulatory molecules,such as the galactosylated oligosaccharides as specified in thisspecification and fatty acids (PUFAs), fish oil, amino acids (e.g.,arginine and glutamine), lectins (e.g., selectins), vitamins (e.g.,vitamins A, B6, C, E, thiamine, folate) and minerals (e.g., zinc). Suchgalactosylated oligosaccharides may include LNB, LacNAc, poly-LacNAc,novo-LNP I, Gal-b1,4-GlcNAc-b1,6-(GlcNAc-b1,3)-Gal-b1,4-Glc, 3-FLN(Gal-b1,4-(Fuc-a1,3)-GlcNAc, SLNPa(Gal-b1,4-GlcNAc-b1,6-(Neu5Ac-a2,3-Gal-b1,3)-Gal-b1,4-Glc), LNT,iso-LNT, novo LNT, Gal-Novo-LNP I, Gal-Novo-LNP II, LNnT, LNnH,DGal-LNnH, Gal-LNFP III, DF DGal-LNnH, DF DGal-LNnT, TF DGal-LNnH a, TFDGal-LNnH b, FS Gal-LNnH, galilipentasaccharide, para-LNnH. In addition,examples of immunomodulatory ingredients present in pharmaceuticalingredients and medicines comprise other immunomodulatory molecules,such as a galactosylated oligosaccharide as specified in thisspecification and interleukins, lipopolysaccharides, glucan, interferongamma and specific antibodies. Such galactosylated oligosaccharidespresent in pharmaceutical mixtures and/or medicines may include LNB,LacNAc, poly-LacNAc, novo-LNP I,Gal-b1,4-GlcNAc-b1,6-(GlcNAc-b1,3)-Gal-b1,4-Glc, 3-FLN(Gal-b1,4-(Fuc-a1,3)-GlcNAc, SLNPa(Gal-b1,4-GlcNAc-b1,6-(Neu5Ac-a2,3-Gal-b1,3)-Gal-b1,4-Glc), LNT,iso-LNT, novo LNT, Gal-Novo-LNP I, Gal-Novo-LNP II, LNnT, LNnH,DGal-LNnH, Gal-LNFP III, DF DGal-LNnH, DF DGal-LNnT, TF DGal-LNnH a, TFDGal-LNnH b, FS Gal-LNnH, galilipentasaccharide, para-LNnH.

Each embodiment disclosed in the context of one aspect of thedisclosure, is also disclosed in the context of all other aspects of thedisclosure, unless explicitly stated otherwise.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this disclosure belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization described above and below are thosewell-known and commonly employed in the art. Standard techniques areused for nucleic acid and peptide synthesis. Generally, purificationsteps are performed according to the manufacturer's specifications.

Further advantages follow from the specific embodiments, the examplesand the attached drawings. It goes without saying that theabovementioned features and the features that are still to be explainedbelow can be used not only in the respectively specified combinations,but also in other combinations or on their own, without departing fromthe scope of the disclosure.

The disclosure relates to the following specific embodiments:

-   -   1. Use of an N-acetylglucosamine b-1,X-galactosyltransferase for        the synthesis of a galactosylated disaccharide or        oligosaccharide, wherein the N-acetylglucosamine        b-1,X-galactosyltransferase galactosylates        -   an N-acetylglucosamine and/or N-acetylgalactosamine as a            monosaccharide, and/or        -   an N-acetylglucosamine and/or N-acetylgalactosamine as part            of a di- and/or oligosaccharide at the non-reducing end of            the di- and/or oligosaccharide,        -   characterized in that the N-acetylglucosamine            b-1,X-galactosyltransferase is:        -   A. an N-acetylglucosamine b-1,3-galactosyltransferase that            has            -   a. PFAM domain PF00535 and                -   i) comprises the sequence [AGPS]XXLN(X_(n))RXDXD                    with SEQ ID NO: 1, wherein X is any amino acid                    except for the combination XX on positions 2 and 3                    that cannot be an FA, FS, YC or YS combination and                    wherein n is 12 to 17, or                -   ii) comprises the sequence PXXLN(X_(n))RXDXD(X_(m))                    [FWY]XX[HKR]XX[NQST] with SEQ ID NO: 2, wherein X is                    any amino acid except for the combination XX on                    positions 2 and 3 that cannot be an FA, FS, YC or YS                    combination and wherein n is 12 to 17 and m is 100                    to 115, or                -   iii) comprises a polypeptide sequence according to                    any one of SEQ ID NOs: 3 or 4, or                -   iv) is a functional homologue, variant or derivative                    of any one of SEQ ID NOs: 3 or 4 having at least 80%                    overall sequence identity to the full length of any                    one of the N-acetylglucosamine                    b-1,3-galactosyltransferase polypeptide with SEQ ID                    NOs: 3 or 4 and having N-acetylglucosamine                    b-1,3-galactosyltransferase activity, or                -   v) comprises an oligopeptide sequence of at least 8,                    9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20                    consecutive amino acid residues from any one of SEQ                    ID NOs: 3 or 4 and having N-acetylglucosamine                    b-1,3-galactosyltransferase activity, or            -   b. PFAM domain 1PR002659 and                -   i) comprises the sequence KT(Xn)[FY]XXKXDXD                    (Xm)[FHY]XXG(X, no A, G, S)(Xp)(X, no F, H, W,                    Y)[DE]D[ILV]XX [AG] with SEQ ID NO: 05, wherein X is                    any amino acid and wherein n is 13 to 16, m is 35 to                    70 and p is 20 to 45, or                -   ii) comprises a polypeptide sequence according to                    any one of SEQ ID NOs: 6, 7, 8 or 9, or                -   iii) is a functional homologue, variant or                    derivative of any one of SEQ ID NOs: 6, 7, 8 or 9                    having at least 80% overall sequence identity to the                    full length of any one of the N-acetylglucosamine                    b-1,3-galactosyltransferase polypeptide with SEQ ID                    NOs: 6, 7, 8 or 9 and having N-acetylglucosamine                    b-1,3-galactosyltransferase activity, or                -   iv) comprises an oligopeptide sequence of at least                    8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20                    consecutive amino acid residues from any one of SEQ                    ID NOs: 6, 7, 8 or 9 and having N-acetylglucosamine                    b-1,3-galactosyltransferase activity, or        -   B. an N-acetylglucosamine b-1,4-galactosyltransferase that            has            -   a. PFAM domain PF01755 and                -   i) comprises the sequence                    EXXCXXSHX[AFILTY]LW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with                    SEQ ID NO: 10, wherein X is any amino acid and                    wherein n is 13 to 15 and m is 50 to 75,                -   ii) comprises the sequence EXXCXXSH[LR]VLW(X_(n))                    EDD(X_(m))[ACGST]XXY[ILMV] with SEQ ID NO: 11,                    wherein X is any amino acid and wherein n is 13 to                    15 and m is 50 to 75,                -   iii) comprises the sequence EXXCXXSH[VHI]SLW                    (X_(n))EDD(X_(m))[ACGST]XXY[ILMV] with SEQ ID NO:                    12, wherein X is any amino acid and wherein n is 13                    to 15 and m is 50 to 75,                -   iv) comprises the sequence EXXCXXSHYMLW(X_(n))                    EDD(X_(m))[ACGST]XXY[ILMV] with SEQ ID NO: 13,                    wherein X is any amino acid and wherein n is 13 to                    15 and m is 50 to 75,                -   v) comprises the sequence EXXCXXSHXX(X, no                    V)Y(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 14,                    wherein X is any amino acid and wherein n is 13 to                    15 and m is 50 to 75,                -   vi) comprises a polypeptide sequence according to                    any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21,                    22 or 23,                -   vii) is a functional homologue, variant or                    derivative of any one of SEQ ID NOs: 15, 16, 17, 18,                    19, 20, 21, 22 or 23 having at least 80% overall                    sequence identity to the full length of any one of                    the N-acetylglucosamine b-1,4-galactosyltransferase                    polypeptide with SEQ ID NOs: 15, 16, 17, 18, 19, 20,                    21, 22 or 23 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or                -   viii) comprises an oligopeptide sequence of at least                    8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20                    consecutive amino acid residues from any one of SEQ                    ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23 and                    having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or            -   b. PFAM domain PF00535 and                -   i) comprises the sequence R[KN]XXXXXXXGXXXX [FL](X,                    no V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE] with SEQ ID NO:                    24 wherein X is any amino acid and wherein n is 50                    to 75 and m is 10 to 30, or                -   ii) comprises the sequence R[KN]XXXXXXXGXXXX [FL](X,                    no                    V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE](Xp)[FWY]XX[HKR]XX[NQST]                    with SEQ ID NO: 25 wherein X is any amino acid and                    wherein n is 50 to 75, m is 10 to 30 and p is 20 to                    25, or                -   iii) comprises a polypeptide sequence according to                    any one of SEQ ID NOs: 26 or 27, or                -   iv) is a functional homologue, variant or derivative                    of any one of SEQ ID NOs: 26 or 27 having at least                    80% overall sequence identity to the full length of                    any one of the N-acetylglucosamine                    b-1,4-galactosyltransferase polypeptide with SEQ ID                    NOs: 26 or 27 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or                -   v) comprises an oligopeptide sequence of at least 8,                    9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20                    consecutive amino acid residues from any one of SEQ                    ID NOs: 26 or 27 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or            -   c. PFAM domain PF02709 and not PFAM domain PF00535, and                -   i) comprises the sequence [FWY]XX[FWY](X_(n))                    [FWY][GQ]X[DE]D with SEQ ID NO: 28 wherein X is any                    amino acid except for the combination XX on                    positions 2 and 3 that cannot be an IP or NL                    combination and wherein n is 21 to 26, or                -   ii) comprises a polypeptide sequence according to                    any one of SEQ ID NOs: 29, 30, 31, 32, 33 or 34, or                -   iii) is a functional homologue, variant or                    derivative of any one of SEQ ID NOs: 29, 30, 31, 32,                    33 or 34 having at least 80% overall sequence                    identity to the full length of any one of the                    N-acetylglucosamine b-1,4-galactosyltransferase                    polypeptide with SEQ ID NOs: 29, 30, 31, 32, 33 or                    34 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or                -   iv) comprises an oligopeptide sequence of at least                    8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20                    consecutive amino acid residues from any one of SEQ                    ID NOs: 29, 30, 31, 32, 33 or 34 and having                    N-acetylglucosamine b-1,4-galactosyltransferase                    activity, or            -   d. PFAM domain PF03808 and                -   i) comprises the sequence [ST][FHY]XN(Xn)DGXXX                    XXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA with SEQ ID NO:                    35, wherein X is any amino acid and wherein n is 20                    to 25, or                -   ii) comprises the sequence [ST][FHY]XN(Xn)DGXXX                    XXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA(Xm)[HR]XG[FW                    Y](Xp)GXGXXXQ[DE] with SEQ ID NO: 36, wherein X is                    any amino acid and wherein n is 20 to 25, m is 40 to                    50 and p is 22 to 30, or                -   iii) comprises a polypeptide sequence according to                    any one of SEQ ID NOs: 37, 38 or 39, or                -   iv) is a functional homologue, variant or derivative                    of any one of SEQ ID NOs: 37, 38 or 39 having at                    least 80% overall sequence identity to the full                    length of any one of the N-acetylglucosamine                    b-1,4-galactosyltransferase polypeptide with SEQ ID                    NOs: 37, 38 or 39 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or                -   v) comprises an oligopeptide sequence of at least 8,                    9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20                    consecutive amino acid residues from any one of SEQ                    ID NOs: 37, 38 or 39 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity.    -   2. A method to synthesize a galactosylated disaccharide or        oligosaccharide by use of an N-acetylglucosamine        b-1,X-galactosyltransferase according to embodiment 1.    -   3. Method according to embodiment 2, wherein the synthesis        comprises the steps of:        -   a. providing UDP-galactose and any one of the            galactosyltransferase, wherein the galactosyltransferase is            capable of transferring a galactose residue from the            UDP-galactose donor to one or more acceptor(s),        -   b. contacting any one of the galactosyltransferase and            UDP-galactose with one or more acceptor(s), under conditions            where the galactosyltransferase catalyzes the transfer of a            galactose residue from the UDP-galactose to the acceptor(s),            and        -   c. preferably, separating the galactosylated di- or            oligosaccharide.    -   4. Method according to embodiment 3, wherein the acceptor(s)        is/are an N-acetylglucosamine and/or an N-acetylgalactosamine as        a monosaccharide, and/or a di- and/or oligosaccharide having an        N-acetylglucosamine and/or N-acetylgalactosamine at its        non-reducing end.    -   5. Method according to any one of embodiments 2 to 4, wherein        the galactosylated disaccharide or oligosaccharide is produced        in a cell-free system.    -   6. Method according to any one of embodiments 2 to 4, wherein        the galactosylated disaccharide or oligosaccharide is produced        by a cell.    -   7. Method according to embodiment 6, wherein the cell        -   is capable of synthesizing one or more of the acceptor(s),            and        -   expresses any one of the N-acetylglucosamine            b-1,3-galactosyltransferases and/or N-acetylglucosamine            b-1,4-galactosyltransferases, and        -   is capable of synthesizing UDP-galactose (UDP-Gal) as donor            for the galactosyltransferases.    -   8. Method according to any one of embodiments 6 or 7, wherein        the cell is further capable of synthesizing one or more        nucleotide-sugar donor(s) chosen from the list comprising:        GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal,        UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine        (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc),        CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), UDP-glucuronate,        UDP-galacturonate, GDP-rhamnose, UDP-xylose.    -   9. Method according to any one of embodiments 6 to 8, wherein        the cell is further capable of expressing one or more        glycosyltransferases selected from the list comprising:        fucosyltransferases, sialyltransferases, galactosyltransferases,        glucosyltransferases, mannosyltransferases,        N-acetylglucosaminyltransferases,        N-acetylgalactosaminyltransferases,        N-acetylmannosaminyltransferases, xylosyltransferases,        glucuronyltransferases, galacturonyltransferases,        glucosaminyltransferases, N-glycolyineuraminyltransferases,        rhamnosyltransferases.    -   10. Method according to any one of embodiments 6 to 9, wherein        the cell is a metabolically engineered cell.    -   11. Method according to any one of embodiments 6 to 10, wherein        the cell is modified in the expression or activity of an enzyme        selected from the group comprising: glucosamine 6-phosphate        N-acetyltransferase, phosphatase, glycosyltransferase,        L-glutanine-D-fructose-6-phosphate aminotransferase, and        UDP-glucose 4-epimerase.    -   12. Method according to any one of embodiments 6 to 11, wherein        the cell is unable to convert N-acetylglucosamine-6-phosphate to        glucosamine-6-phosphate, and/or unable to convert        glucosamine-6-phosphate to fructose-6-phosphate.    -   13. Method according to any one of embodiments 6 to 12, wherein        the cell is modified for enhanced UDP-galactose production and        wherein the modification is chosen from the group comprising:        knock-out of a 5′-nucleotidase/UDP-sugar hydrolase encoding gene        or knock-out of a galactose-1-phosphate uridylyltransferase        encoding gene.    -   14. Method according to any one of embodiments 6 to 13, wherein        the cell is capable of catabolizing a carbon source selected        from the list comprising glucose, fructose, mannose, galactose,        lactose, sucrose, maltose, malto-oligosaccharides, trehalose,        starch, cellulose, hemi-cellulose, corn-steep liquor,        high-fructose syrup, glycerol, acetate, citrate, lactate and        pyruvate.    -   15. The method according to any one of embodiments 3 to 14,        wherein the separation comprises at least one of the following        steps: clarification, ultrafiltration, nanofiltration, reverse        osmosis, microfiltration, activated charcoal or carbon        treatment, tangential flow high-performance filtration,        tangential flow ultrafiltration, affinity chromatography, ion        exchange chromatography, hydrophobic interaction chromatography        and/or gel filtration, ligand exchange chromatography.    -   16. The method according to any one of embodiments 3 to 15,        further comprising purification of the galactosylated di- or        oligosaccharide.    -   17. The method according to embodiment 16, wherein the        purification comprises at least one of the following steps: use        of activated charcoal or carbon, use of charcoal,        nanofiltration, ultrafiltration or ion exchange, use of        alcohols, use of aqueous alcohol mixtures, crystallization,        evaporation, precipitation, drying, spray drying or        lyophilization.    -   18. A cell metabolically engineered to synthesize a        galactosylated disaccharide or oligosaccharide by use of an        N-acetylglucosamine b-1,X-galactosyltransferase according to        embodiment 1.    -   19. Cell according to embodiment 18, wherein the cell        -   expresses any one of the N-acetylglucosamine            b-1,3-galactosyltransferases and/or N-acetylglucosamine            b-1,4-galactosyltransferases,        -   is capable of synthesizing UDP-galactose (UDP-Gal) as donor            for the galactosyltransferases, and        -   is capable of synthesizing one or more acceptor(s) for the            galactosyltransferases, wherein the acceptor(s) is/are an            N-acetylglucosamine as a monosaccharide, and/or a di- or            oligosaccharide having an N-acetylglucosamine and/or            N-acetylgalactosamine at its non-reducing end.    -   20. Cell according to any one of embodiments 18 or 19, wherein        the cell is further capable of synthesizing one or more        nucleotide-sugar donor(s) chosen from the list comprising:        GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal,        UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine        (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc),        CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), UDP-glucuronate,        UDP-galacturonate, GDP-rhamnose, UDP-xylose.    -   21. Cell according to any one of embodiments 18 to 20, wherein        the cell is further capable of expressing one or more        glycosyltransferases selected from the list comprising:        fucosyltransferases, sialyltransferases, galactosyltransferases,        glucosyltransferases, mannosyltransferases,        N-acetylglucosaminyltransferases,        N-acetylgalactosaminyltransferases,        N-acetylmannosaminyltransferases, xylosyltransferases,        glucuronyltransferases, galacturonyltransferases,        glucosaminyltransferases, N-glycolylneuraminyltransferases,        rhamnosyltransferases.    -   22. Cell according to any one of embodiments 18 to 21, wherein        the cell is modified in the expression or activity of an enzyme        selected from the group comprising: glucosamine 6-phosphate        N-acetyltransferase, phosphatase, glycosyltransferase,        L-glutamine D-fructose-6-phosphate aminotransferase, and        UDP-glucose 4-epimerase.    -   23. Cell according to any one of embodiments 18 to 22, wherein        the cell is unable to convert N-acetylglucosamine-6-phosphate to        glucosamine-6-phosphate, and/or unable to convert        glucosamine-6-phosphate to fructose-6-phosphate.    -   24. Cell according to any one of embodiments 18 to 23, wherein        the cell is modified for enhanced UDP-galactose production and        wherein the modification is chosen from the group comprising:        knock-out of a 5′-nucleotidase/UDP-sugar hydrolase encoding gene        or knock-out of a galactose-1-phosphate uridylyltransferase        encoding gene.    -   25. Cell according to any one of embodiments 18 to 24, wherein        the cell is capable of catabolizing a carbon source selected        from the list comprising glucose, fructose, mannose, galactose,        lactose, sucrose, maltose, malto-oligosaccharides, trehalose,        starch, cellulose, hemi-cellulose, corn-steep liquor,        high-fructose syrup, glycerol, acetate, citrate, lactate and        pyruvate.    -   26. The cell according to any one of embodiments 18 to 25 or        method according to any one of embodiments 3 to 17, wherein the        cell is selected from the group consisting of microorganism,        plant, or animal cells, preferably the microorganism is a        bacterium, fungus or a yeast, preferably the plant is a rice,        cotton, rapeseed, soy, maize or corn plant, preferably the        animal is an insect, fish, bird or non-human mammal, preferably        the animal cell is a mammalian cell line.    -   27. The cell according to any one of embodiments 18 to 26 or        method according to any one of embodiments 3 to 17 and 26,        wherein the cell is a cell of a bacterium, preferably of an        Escherichia coli strain, more preferably of an Escherichia coli        strain, which is a K-12 strain, even more preferably the        Escherichia coli K-12 strain is 1K coil MG1655.    -   28. The cell according to any one of embodiments 18 to 26 or        method according to any one of embodiments 3 to 17 and 26,        wherein the cell is a yeast cell.

Moreover, the disclosure relates to the following preferred specificembodiments:

-   -   1. Use of an N-acetylglucosamine b-1,X-galactosyltransferase for        the synthesis of a galactosylated disaccharide or        oligosaccharide, wherein the N-acetylglucosamine        b-1,X-galactosyltransferase galactosylates        -   an N-acetylglucosamine and/or N-acetylgalactosamine as a            monosaccharide, and/or        -   an N-acetylglucosamine and/or N-acetylgalactosamine as part            of a di- and/or oligosaccharide at the non-reducing end of            the di- and/or oligosaccharide,        -   characterized in that the N-acetylglucosamine            b-1,X-galactosyltransferase is:        -   A. an N-acetylglucosamine b-1,3-galactosyltransferase that            has            -   a. PFAM domain PF00535 and                -   i) comprises the sequence [AGPS]XXLN(X_(n))RXDXD                    with SEQ ID NO: 1, wherein X is any amino acid                    except for the combination XX on positions 2 and 3                    that cannot be an FA, FS, YC or YS combination and                    wherein n is 12 to 17, or                -   ii) comprises the sequence PXXLN(X_(n))RXDXD(X_(m))                    [FWY]XX[HKR]X[NQST] with SEQ ID NO: 2, wherein X is                    any amino acid except for the combination XX on                    positions 2 and 3 that cannot be an FA, FS, YC or YS                    combination and wherein n is 12 to 17 and m is 100                    to 115, or                -   iii) comprises a polypeptide sequence according to                    any one of SEQ ID NOs: 3 or 4, or                -   iv) is a functional homologue, variant or derivative                    of any one of SEQ ID NOs: 3 or 4 having at least 80%                    overall sequence identity to the full length of any                    one of the N-acetylglucosamine                    b-1,3-galactosyltransferase polypeptide with SEQ ID                    NOs: 3 or 4 and having N-acetylglucosamine                    b-1,3-galactosyltransferase activity, or                -   v) comprises an oligopeptide sequence of at least 8,                    9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20                    consecutive amino acid residues from any one of SEQ                    ID NOs: 3 or 4 and having N-acetylglucosamine                    b-1,3-galactosyltransferase activity, or                -   vi) is a functional fragment of any one of SEQ ID                    NOs: 3 or 4 and having N-acetylglucosamine                    b-1,3-galactosyltransferase activity, or                -   vii) comprises a polypeptide comprising or                    consisting of an amino acid sequence having at least                    80% sequence identity to the full-length amino acid                    sequence of any one of SEQ ID NOs: 3 or 4 and having                    N-acetylglucosamine b-1,3-galactosyltransferase                    activity, or            -   b. PFAM domain 1PR002659 and                -   i) comprises the sequence KT(Xn)[FY]XXKXDXD                    (Xm)[FHY]XXG(X, no A, G, S)(Xp)(X, no F, H, W,                    Y)[DE]D[ILV]XX[AG] with SEQ ID NO: 05, wherein X is                    any amino acid and wherein n is 13 to 16, m is 35 to                    70 and p is 20 to 45, or                -   ii) comprises a polypeptide sequence according to                    any one of SEQ ID NOs: 6, 7, 8 or 9, or                -   iii) is a functional homologue, variant or                    derivative of any one of SEQ ID NOs: 6, 7, 8 or 9                    having at least 80% overall sequence identity to the                    full length of any one of the N-acetylglucosamine                    b-1,3-galactosyltransferase polypeptide with SEQ ID                    NOs: 6, 7, 8 or 9 and having N-acetylglucosamine                    b-1,3-galactosyltransferase activity, or                -   iv) comprises an oligopeptide sequence of at least                    8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20                    consecutive amino acid residues from any one of SEQ                    ID NOs: 6, 7, 8 or 9 and having N-acetylglucosamine                    b-1,3-galactosyltransferase activity, or                -   v) is a functional fragment of any one of SEQ ID                    NOs: 6, 7, 8 or 9 and having N-acetylglucosamine                    b-1,3-galactosyltransferase activity, or                -   vi) comprises a polypeptide comprising or consisting                    of an amino acid sequence having at least 80%                    sequence identity to the full-length amino acid                    sequence of any one of SEQ ID NOs: 6, 7, 8 or 9 and                    having N-acetylglucosamine                    b-1,3-galactosyltransferase activity, or        -   B. an N-acetylglucosamine b-1,4-galactosyltransferase that            has            -   a. PFAM domain PF01755 and                -   i) comprises the sequence                    EXXCXXSHX[AFILTY]LW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with                    SEQ ID NO: 10, wherein X is any amino acid and                    wherein n is 13 to 15 and m is 50 to 75, or                -   ii) comprises the sequence EXXCXXSH[LR]VLW(X_(n))                    EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 11, wherein                    X is any amino acid and wherein n is 13 to 15 and m                    is 50 to 75, or                -   iii) comprises the sequence EXXCXXSH[VHI]SLW                    (X_(n))EDD(X_(m))[ACGST]XXY[ILMV] with SEQ ID NO:                    12, wherein X is any amino acid and wherein n is 13                    to 15 and m is 50 to 75, or                -   iv) comprises the sequence EXXCXXSHYMLW(X_(n))                    EDD(X_(m))[ACGST]XXY[ILMV] with SEQ ID NO: 13,                    wherein X is any amino acid and wherein n is 13 to                    15 and m is 50 to 75, or                -   v) comprises the sequence EXXCXXSHXX(X, no V)Y                    (Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 14,                    wherein X is any amino acid and wherein n is 13 to                    15 and m is 50 to 75, or                -   vi) comprises a polypeptide sequence according to                    any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21,                    22 or 23, or                -   vii is a functional homologue, variant or derivative                    of any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20,                    21, 22 or 23 having at least 80% overall sequence                    identity to the full length of any one of the                    N-acetylglucosamine b-1,4-galactosyltransferase                    polypeptide with SEQ ID NOs: 15, 16, 17, 18, 19, 20,                    21, 22 or 23 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or                -   viii) comprises an oligopeptide sequence of at least                    8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20                    consecutive amino acid residues from any one of SEQ                    ID NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23 and                    having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or                -   ix) is a functional fragment of any one of SEQ ID                    NOs: 15, 16, 17, 18, 19, 20, 21, 22 or 23 and having                    N-acetylglucosamine b-1,4-galactosyltransferase                    activity, or                -   x) comprises a polypeptide comprising or consisting                    of an amino acid sequence having at least 80%                    sequence identity to the full-length amino acid                    sequence of any one of SEQ ID NOs: 15, 16, 17, 18,                    19, 20, 21, 22 or 23 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or            -   b. PFAM domain PF00535 and                -   i) comprises the sequence R[KN]XXXXXXXGXXX X[FL](X,                    no V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE] with SEQ ID NO:                    24 wherein X is any amino acid and wherein n is 50                    to 75 and m is 10 to 30, or                -   ii) comprises the sequence R[KN]XXXXXXXGXXX X[FL](X,                    no                    V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE](Xp)[FWY]XX[HKR]XX[NQST]                    with SEQ ID NO: 25 wherein X is any amino acid and                    wherein n is 50 to 75, m is 10 to 30 and p is 20 to                    25, or                -   iii) comprises a polypeptide sequence according to                    any one of SEQ ID NOs: 26 or 27, or                -   iv) is a functional homologue, variant or derivative                    of any one of SEQ ID NOs: 26 or 27 having at least                    80% overall sequence identity to the full length of                    any one of the N-acetylglucosamine                    b-1,4-galactosyltransferase polypeptide with SEQ ID                    NOs: 26 or 27 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or                -   v) comprises an oligopeptide sequence of at least 8,                    9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20                    consecutive amino acid residues from any one of SEQ                    ID NOs: 26 or 27 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or                -   vi) is a functional fragment of any one of SEQ ID                    NOs: 26 or 27 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or                -   vii) comprises a polypeptide comprising or                    consisting of an amino acid sequence having at least                    80% sequence identity to the full-length amino acid                    sequence of any one of SEQ ID NOs: 26 or 27 and                    having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or            -   c. PFAM domain PF02709 and not PFAM domain PF00535, and                -   i) comprises the sequence [FWY]XX[FWY](X_(n))                    [FWY][GQ]X[DE]D with SEQ ID NO: 28 wherein X is any                    amino acid except for the combination XX on                    positions 2 and 3 that cannot be an IP or NL                    combination and wherein n is 21 to 26, or                -   ii) comprises a polypeptide sequence according to                    any one of SEQ ID NOs: 29, 30, 31, 32, 33 or 34, or                -   iii) is a functional homologue, variant or                    derivative of any one of SEQ ID NOs: 29, 30, 31, 32,                    33 or 34 having at least 80% overall sequence                    identity to the full length of any one of the                    N-acetylglucosamine b-1,4-galactosyltransferase                    polypeptide with SEQ ID NOs: 29, 30, 31, 32, 33 or                    34 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or                -   iv) comprises an oligopeptide sequence of at least                    8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20                    consecutive amino acid residues from any one of SEQ                    ID NOs: 29, 30, 31, 32, 33 or 34 and having                    N-acetylglucosamine b-1,4-galactosyltransferase                    activity, or                -   v) is a functional fragment of any one of SEQ ID                    NOs: 29, 30, 31, 32, 33 or 34 and having                    N-acetylglucosamine b-1,4-galactosyltransferase                    activity, or                -   vi) comprises a polypeptide comprising or consisting                    of an amino acid sequence having at least 80%                    sequence identity to the full-length amino acid                    sequence of any one of SEQ ID NOs: 29, 30, 31, 32,                    33 or 34 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or            -   d. PFAM domain PF03808 and                -   i) comprises the sequence [ST][FHY]XN(Xn)DGXX                    XXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA with SEQ ID NO:                    35, wherein X is any amino acid and wherein n is 20                    to 25, or                -   ii) comprises the sequence [ST][FHY]XN(Xn)DGXXX                    XXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA(Xm)[HR]XG[FW                    Y](Xp)GXGXXXQ[DE] with SEQ ID NO: 36, wherein X is                    any amino acid and wherein n is 20 to 25, m is 40 to                    50 and p is 22 to 30, or                -   iii) comprises a polypeptide sequence according to                    any one of SEQ ID NOs: 37, 38 or 39, or                -   iv) is a functional homologue, variant or derivative                    of any one of SEQ ID NOs: 37, 38 or 39 having at                    least 80% overall sequence identity to the full                    length of any one of the N-acetylglucosamine                    b-1,4-galactosyltransferase polypeptide with SEQ ID                    NOs: 37, 38 or 39 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or                -   v) comprises an oligopeptide sequence of at least 8,                    9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20                    consecutive amino acid residues from any one of SEQ                    ID NOs: 37, 38 or 39 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or                -   vi) functional fragment of any one of SEQ ID NOs:                    37, 38 or 39 and having N-acetylglucosamine                    b-1,4-galactosyltransferase activity, or                -   ii) comprises a polypeptide comprising or consisting                    of an amino acid sequence having at least 80%                    sequence identity to the full-length amino acid                    sequence of any one of SEQ ID NOs: 37, 38 or 39 and                    having N-acetylglucosamine                    b-1,4-galactosyltransferase activity.    -   2. A method to synthesize a galactosylated disaccharide or        oligosaccharide by use of an N-acetylglucosamine        b-1,X-galactosyltransferase according to preferred embodiment 1.    -   3. Method according to preferred embodiment 2, wherein the        synthesis comprises the steps of:        -   a. providing UDP-galactose and any one of the            galactosyltransferase, wherein the galactosyltransferase is            capable of transferring a galactose residue from the            UDP-galactose donor to one or more acceptor(s), and        -   b. contacting any one of the galactosyltransferase and            UDP-galactose with one or more acceptor(s), under conditions            where the galactosyltransferase catalyzes the transfer of a            galactose residue from the UDP-galactose to the acceptor(s),        -   c. preferably, separating the galactosylated di- or            oligosaccharide.    -   4. Method according to preferred embodiment 3, wherein the        acceptor(s) is/are an N-acetylglucosamine and/or an        N-acetylgalactosamine as a monosaccharide, and/or a di- and/or        oligosaccharide having an N-acetylglucosamine and/or        N-acetylgalactosamine at its non-reducing end.    -   5. Method according to any one of preferred embodiments 2 to 4,        wherein the galactosylated disaccharide or oligosaccharide is        produced in a cell-free system,    -   6. Method according to any one of preferred embodiments 2 to 4,        wherein the galactosylated disaccharide or oligosaccharide is        produced by a cell.    -   7. Method according to preferred embodiment 6, wherein the cell        -   is capable of synthesizing one or more of the acceptor(s),            and        -   expresses any one of the N-acetylglucosamine            b-1,3-galactosyltransferases and/or N-acetylglucosamine            b-1,4-galactosyltransferases, and        -   is capable of synthesizing UDP-galactose (UDP-Gal) as donor            for the galactosyltransferases.    -   8. Method according to any one of specific embodiments 6 or 7,        wherein the cell is further capable of synthesizing one or more        nucleotide-sugar donor(s) chosen from the list comprising:        GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal,        UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine        (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc),        UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,        UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,        UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or        UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,        UDP-N-acetylfucosamine (UDP-L-FucNAc or        UDP-2-acetamido-2,6-dideoxy-L-galactose),        UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or        UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid,        UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or        UDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose,        C1P—N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac,        CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2,        CMP-Neu5,7(8,9)Ac2, UDP-glucuronate, UDP-galacturonate,        GDP-rhamnose, UDP-xylose.    -   9. Method according to any one of preferred embodiments 6 to 8,        wherein the cell is further capable of expressing one or more        glycosyltransferases selected from the list comprising:        fucosyltransferases, sialyltransferases, galactosyltransferases,        glucosyltransferases, mannosyltransferases,        N-acetylglucosaminyltransferases,        N-acetylgalactosaminyltransferases,        N-acetylmannosaminyltransferases, xylosyltransferases,        glucuronyltransferases, galacturonyltransferases,        glucosaminyltransferases, N-glycolylneuraminyltransferases,        rhamnosyltransferases, N-acetylrhamnosyltransferases,        UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine        transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases        and fucosaminyltransferases,        -   preferably, the fucosyltransferase is chosen from the list            comprising alpha-1,2-fucosyltransferase,            alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase            and alpha-1,6-fucosyltransferase,        -   preferably, the sialyltransferase is chosen from the list            comprising alpha-2,3-sialyltransferase,            alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase,        -   preferably, the galactosyltransferase is chosen from the            list comprising beta-1,3-galactosyltransferase,            N-acetylglucosamine beta-1,3-galactosyltransferase,            beta-1,4-galactosyltransferase, N-acetylglucosamine            beta-1,4-galactosyltransferase,            alpha-1,3-galactosyltransferase and            alpha-1,4-galactosyltransferase,        -   preferably, the glucosyltransferase is chosen from the list            comprising alpha-glucosyltransferase,            beta-1,2-glucosyltransferase, beta-1,3-glucosyltransferase            and beta-1,4-glucosyltransferase,        -   preferably, the mannosyltransferase is chosen from the list            comprising alpha-1,2-mannosyltransferase,            alpha-1,3-mannosyltransferase and            alpha-1,6-mannosyltransferase,        -   preferably, the N-acetylglucosaminyltransferase is chosen            from the list comprising galactoside            beta-1,3-N-acetylglucosaminyltransferase and            beta-1,6-N-acetylglucosaminyltransferase,        -   preferably, the N-acetylgalactosaminyltransferase is an            alpha-1,3-N-acetylgalactosaminyltransferase.    -   10. Method according to any one of preferred embodiments 6 to 9,        wherein the cell is a metabolically engineered cell.    -   11. The method according to preferred embodiment 10, wherein the        cell is modified with one or more gene expression modules,        characterized in that the expression from any of the expression        modules is either constitutive or is created by a natural        inducer.    -   12. The method according to any one of preferred embodiment 10        or 11, wherein the cell comprises multiple copies of the same        coding DNA sequence encoding for one protein.    -   13. Method according to any one of preferred embodiments 6 to        12, wherein the cell is modified in the expression or activity        of an enzyme selected from the group comprising: glucosamine        6-phosphate N-acetyltransferase, phosphatase,        glycosyltransferase, L-glutanine-D-fructose-6-phosphate        aminotransferase, and UDP-glucose 4-epimerase.    -   14. Method according to any one of preferred embodiments 6 to        13, wherein the cell is unable to convert        N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate,        and/or unable to convert glucosamine-6-phosphate to        fructose-6-phosphate.    -   15. Method according to any one of preferred embodiments 6 to        14, wherein the cell is modified for enhanced UDP-galactose        production and wherein the modification is chosen from the group        comprising: knock-out of a 5′-nucleotidase/UDP-sugar hydrolase        encoding gene or knock-out of a galactose-1-phosphate        uridylyltransferase encoding gene.    -   16. Method according to any one of preferred embodiments 6 to        15, wherein the cell is using one or more precursor(s) for the        production of the galactosylated disaccharide or        oligosaccharide, the precursor(s) being fed to the cell from the        cultivation medium.    -   17. Method according to any one of preferred embodiments 6 to        16, wherein the cell is producing one or more precursor(s) for        the production of the galactosylated disaccharide or        oligosaccharide.    -   18. Method according to any one of preferred embodiments 16 or        17, wherein the precursor for the production of the        galactosylated disaccharide or oligosaccharide is completely        converted into the galactosylated disaccharide or        oligosaccharide.    -   19. Method according to any one of preferred embodiments 6 to        18, wherein the cell produces the galactosylated disaccharide or        oligosaccharide intracellularly and wherein a fraction or        substantially all of the produced galactosylated disaccharide or        oligosaccharide remains intracellularly and/or is excreted        outside the cell via passive or active transport.    -   20. Method according to any one of preferred embodiments 6 to        19, wherein the cell expresses a membrane transporter protein or        a polypeptide having transport activity hereby transporting        compounds across the outer membrane of the cell wall,        -   preferably, the cell is modified in the expression or            activity of the membrane transporter protein or polypeptide            having transport activity.    -   21. Method according to preferred embodiment 20, wherein the        membrane transporter protein or polypeptide having transport        activity is chosen from the list comprising porters,        P—P-bond-hydrolysis-driven transporters, β-barrel porins,        auxiliary transport proteins, putative transport proteins and        phosphotransfer-driven group translocators,        -   preferably, the porters comprise MFS transporters, sugar            efflux transporters and siderophore exporters,        -   preferably, the P—P-bond-hydrolysis-driven transporters            comprise ABC transporters and siderophore exporters.    -   22. Method according to any one of preferred embodiments 20 or        21, wherein the membrane transporter protein or polypeptide        having transport activity controls the flow over the outer        membrane of the cell wall of the galactosylated disaccharide or        oligosaccharide and/or of one or more precursor(s) and/or        acceptor(s) to be used in the production of the galactosylated        disaccharide or oligosaccharide.    -   23. Method according to any one of preferred embodiments 20 to        22, wherein the membrane transporter protein or polypeptide        having transport activity provides improved production and/or        enabled and/or enhanced efflux of the galactosylated        disaccharide or oligosaccharide.    -   24. Method according to any one of the preferred embodiments 6        to 23, wherein the cell comprises a modification for reduced        production of acetate compared to a non-modified progenitor.    -   25. Method according to preferred embodiment 24, wherein the        cell comprises a lower or reduced expression and/or abolished,        impaired, reduced or delayed activity of any one or more of the        proteins comprising beta-galactosidase, galactoside        O-acetyltransferase, N-acetylglucosamine-6-phosphate        deacetylase, glucosamine-6-phosphate deaminase,        N-acetylglucosamine repressor, ribonucleotide monophosphatase,        EIICBA-Nag, UDP-glucose:undecaprenyl-phosphate        glucose-1-phosphate transferase, L_-fuculokinase, L-fucose        isomerase, N-acetylneuraminate lyase, N-acetylmannosamine        kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man,        EIIC-Man, EIID-Man, ushA, galactose-1-phosphate        uridylyltransferase, glucose-1-phosphate adenylyltransferase,        glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase        isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2,        glucose-6-phosphate isomnerase, aerobic respiration control        protein, transcriptional repressor IclR, Ion protease,        glucose-specific translocating phosphotransferase enzyme IIBC        component ptsG, glucose-specific translocating        phosphotransferase (PTS) enzyme IIBC component malX, enzyme        IIAGlc, beta-glucoside specific PTS enzyme II, fructose-specific        PTS multiphosphoryl transfer protein FruA and FruB, ethanol        dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase,        acetate kinase, phosphoacyltransferase, phosphate        acetyltransferase, pyruvate decarboxylase compared to a        non-modified progenitor.    -   26. Method according to any one of the preferred embodiments 6        to 25, wherein the cell is capable of producing        phosphoenolpyruvate (PEP).    -   27. Method according to any one of the preferred embodiments 6        to 26, wherein the cell is modified for enhanced production        and/or supply of phosphoenolpyruvate (PEP) compared to a        non-modified progenitor.    -   28. Method according to any one of the preferred embodiments 6        to 27, wherein the cell comprises a catabolic pathway for        selected mono-, di- or oligosaccharides that is at least        partially inactivated, the mono-, di-, or oligosaccharides being        involved in and/or required for the production of the        galactosylated disaccharide or oligosaccharide.    -   29. Method according to any one of the preferred embodiments 6        to 28, wherein the cell resists the phenomenon of lactose        killing when grown in an environment in which lactose is        combined with one or more other carbon source(s).    -   30. Method according to any one of the preferred embodiments 6        to 29, wherein the cell produces 90 g/L or more of the        galactosylated disaccharide or oligosaccharide in the whole        broth and/or supernatant and/or wherein the galactosylated        disaccharide or oligosaccharide in the whole broth and/or        supernatant has a purity of at least 80% measured on the total        amount of the galactosylated disaccharide or oligosaccharide and        its precursor(s) in the whole broth and/or supernatant,        respectively.    -   31. Method according to any one of the preferred embodiments 6        to 30, wherein the cell is stably cultured in a medium.    -   32. Method according to any one of the preferred embodiments 6        to 31, wherein the conditions comprise:        -   use of a culture medium comprising at least one precursor            and/or acceptor for the production of the galactosylated            disaccharide or oligosaccharide, and/or        -   adding to the culture medium at least one precursor and/or            acceptor feed for the production of the galactosylated            disaccharide or oligosaccharide.    -   33. Method according to any one of the preferred embodiments 6        to 32, the method comprising at least one of the following        steps:        -   i) Use of a culture medium comprising at least one precursor            and/or acceptor;        -   ii) Adding to the culture medium in a reactor at least one            precursor and/or acceptor feed wherein the total reactor            volume ranges from 250 mL (milliliter) to 10,000 m³ (cubic            meter), preferably in a continuous manner, and preferably so            that the final volume of the culture medium is not more than            three-fold, preferably not more than two-fold, more            preferably less than two-fold of the volume of the culture            medium before the addition of the precursor and/or acceptor            feed;        -   iii) Adding to the culture medium in a reactor at least one            precursor and/or acceptor feed wherein the total reactor            volume ranges from 250 mL (milliliter) to 10,000 m³ (cubic            meter), preferably in a continuous manner, and preferably so            that the final volume of the culture medium is not more than            three-fold, preferably not more than two-fold, more            preferably less than two-fold of the volume of the culture            medium before the addition of the precursor and/or acceptor            feed and wherein preferably, the pH of the precursor and/or            acceptor feed is set between 3 and 7 and wherein preferably,            the temperature of the precursor and/or acceptor feed is            kept between 20° C. and 80° C.;        -   iv) Adding at least one precursor and/or acceptor feed in a            continuous manner to the culture medium over the course of 1            day, 2 days, 3 days, 4 days, 5 days by means of a feeding            solution;        -   v) Adding at least one precursor and/or acceptor feed in a            continuous manner to the culture medium over the course of 1            day, 2 days, 3 days, 4 days, 5 days by means of a feeding            solution and wherein preferably, the pH of the feeding            solution is set between 3 and 7 and wherein preferably, the            temperature of the feeding solution is kept between 20° C.            and 80° C.;        -   the method resulting in the galactosylated disaccharide or            oligosaccharide with a concentration of at least 50 g/L,            preferably at least 75 g/L, more preferably at least 90 g/L,            more preferably at least 100 g/L, more preferably at least            125 g/L, more preferably at least 150 g/L, more preferably            at least 175 g/L, more preferably at least 200 g/L in the            final cultivation.    -   34. Method according to any one of the preferred embodiments 3        to 32, the method comprising at least one of the following        steps:        -   i) Use of a culture medium comprising at least 50, more            preferably at least 75, more preferably at least 100, more            preferably at least 120, more preferably at least 150 gram            of lactose per liter of initial reactor volume wherein the            reactor volume ranges from 250 mL to 10,000 m³ (cubic            meter);        -   ii) Adding to the culture medium a lactose feed comprising            at least 50, more preferably at least 75, more preferably at            least 100, more preferably at least 120, more preferably at            least 150 gram of lactose per liter of initial reactor            volume wherein the reactor volume ranges from 250 mL to            10,000 m³ (cubic meter), preferably in a continuous manner,            and preferably so that the final volume of the culture            medium is not more than three-fold, preferably not more than            two-fold, more preferably less than two-fold of the volume            of the culture medium before the addition of the lactose            feed;        -   iii) Adding to the culture medium a lactose feed comprising            at least 50, more preferably at least 75, more preferably at            least 100, more preferably at least 120, more preferably at            least 150 gram of lactose per liter of initial reactor            volume wherein the reactor volume ranges from 250 mL to            10,000 m³ (cubic meter), preferably in a continuous manner,            and preferably so that the final volume of the culture            medium is not more than three-fold, preferably not more than            two-fold, more preferably less than two-fold of the volume            of the culture medium before the addition of the lactose            feed and wherein preferably the pH of the lactose feed is            set between 3 and 7 and wherein preferably the temperature            of the lactose feed is kept between 20° C. and 80° C.;        -   iv) Adding a lactose feed in a continuous manner to the            culture medium over the course of 1 day, 2 days, 3 days, 4            days, 5 days by means of a feeding solution;        -   v) Adding a lactose feed in a continuous manner to the            culture medium over the course of 1 day, 2 days, 3 days, 4            days, 5 days by means of a feeding solution and wherein the            concentration of the lactose feeding solution is 50 g/L,            preferably 75 g/L, more preferably 100 g/L, more preferably            125 g/L, more preferably 150 g/L, more preferably 175 g/L,            more preferably 200 g/L, more preferably 225 g/L, more            preferably 250 g/L, more preferably 275 g/L, more preferably            300 g/L, more preferably 325 g/L, more preferably 350 g/L,            more preferably 375 g/L, more preferably, 400 g/L, more            preferably 450 g/L, more preferably 500 g/L, even more            preferably, 550 g/L, most preferably 600 g/L; and wherein            preferably the pH of the feeding solution is set between 3            and 7 and wherein preferably the temperature of the feeding            solution is kept between 20° C. and 80° C.;        -   the method resulting in the galactosylated disaccharide or            oligosaccharide with a concentration of at least 50 g/L,            preferably at least 75 g/L, more preferably at least 90 g/L,            more preferably at least 100 g/L, more preferably at least            125 g/L, more preferably at least 150 g/L, more preferably            at least 175 g/L, more preferably at least 200 g/L in the            final volume of the cultivation.    -   35. Method according to preferred embodiment 34, wherein the        lactose feed is accomplished by adding lactose from the        beginning of the cultivation in a concentration of at least 5        mM, preferably in a concentration of 30, 40, 50, 60, 70, 80, 90,        100, 150 mM, more preferably in a concentration >300 mM.    -   36. Method according to any one of preferred embodiment 34 or        35, wherein the lactose feed is accomplished by adding lactose        to the cultivation in a concentration, such, that throughout the        production phase of the cultivation a lactose concentration of        at least 5 mM, preferably 10 mM or 30 mM is obtained.    -   37. Method according to any one of preferred embodiments 6 to        36, wherein the cells are cultivated for at least about 60, 80,        100, or about 120 hours or in a continuous manner.    -   38. Method according to any one of preferred embodiments 6 to        37, wherein the culture medium contains at least one precursor        selected from the group comprising lactose, galactose, fucose        and sialic acid.    -   39. Method according to any one of preferred embodiments 6 to        38, wherein a first phase of exponential cell growth is provided        by adding a carbon-based substrate, preferably glucose or        sucrose, to the culture medium comprising a precursor, followed        by a second phase wherein only a carbon-based substrate,        preferably glucose or sucrose, is added to the culture medium.    -   40. Method according to any one of preferred embodiments 6 to        38, wherein a first phase of exponential cell growth is provided        by adding a carbon-based substrate, preferably glucose or        sucrose, to the culture medium comprising a precursor, followed        by a second phase wherein a carbon-based substrate, preferably        glucose or sucrose, and a precursor are added to the culture        medium.    -   41. Method according to any one of preferred embodiments 6 to        40, wherein the cell is capable of catabolizing a carbon source        selected from the list comprising glucose, fructose, mannose,        galactose, lactose, sucrose, maltose, malto-oligosaccharides,        trehalose, starch, cellulose, hemi-cellulose, corn-steep liquor,        molasses, high-fructose syrup, glycerol, acetate, citrate,        lactate and pyruvate.    -   42. Method according to any one of preferred embodiments 1 to 5,        wherein the method produces a mixture of charged and/or neutral        di- and/or oligosaccharides comprising at least one        galactosylated disaccharide or oligosaccharide.    -   43. Method according to any one of preferred embodiments 1 to 5,        wherein the method produces a mixture of charged and/or neutral        oligosaccharides comprising at least one galactosylated        oligosaccharide.    -   44. Method according to any one of preferred embodiments 6 to        41, wherein the cell produces a mixture of charged, preferably        sialylated, and/or neutral di- and/or oligosaccharides        comprising at least one galactosylated disaccharide or        oligosaccharide.    -   45. Method according to any one of preferred embodiments 6 to        41, wherein the cell produces a mixture of charged, preferably        sialylated, and/or neutral oligosaccharides comprising at least        one galactosylated oligosaccharide.    -   46. Method according to any one of preferred embodiments 3 to        45, wherein the separation comprises at least one of the        following steps: clarification, ultrafiltration, nanofiltration,        two-phase partitioning, reverse osmosis, microfiltration,        activated charcoal or carbon treatment, treatment with non-ionic        surfactants, enzymatic digestion, tangential flow        high-performance filtration, tangential flow ultrafiltration,        affinity chromatography, ion exchange chromatography,        hydrophobic interaction chromatography and/or gel filtration,        ligand exchange chromatography.    -   47. Method according to any one of preferred embodiments 3 to        46, further comprising purification of the galactosylated di- or        oligosaccharide.    -   48. Method according to preferred embodiment 47, wherein the        purification comprises at least one of the following steps: use        of activated charcoal or carbon, use of charcoal,        nanofiltration, ultrafiltration, electrophoresis, enzymatic        treatment or ion exchange, use of alcohols, use of aqueous        alcohol mixtures, crystallization, evaporation, precipitation,        drying, spray drying, lyophilization, spray freeze drying,        freeze spray drying, band drying, belt drying, vacuum band        drying, vacuum belt drying, drum drying, roller drying, vacuum        drum drying or vacuum roller drying.    -   49. A cell metabolically engineered to synthesize a        galactosylated disaccharide or oligosaccharide by use of an        N-acetylglucosamine b-1,X-galactosyltransferase according to        embodiment 1.    -   50. The cell of preferred embodiment 49, wherein the cell        -   expresses any one of the N-acetylglucosamine            b-1,3-galactosyltransferases and/or N-acetylglucosamine            b-1,4-galactosyltransferases, and        -   is capable of synthesizing UDP-galactose (UDP-Gal) as donor            for the galactosyltransferases, and        -   is capable of synthesizing one or more acceptor(s) for the            galactosyltransferases, wherein the acceptor(s) is/are an            N-acetylglucosamine as a monosaccharide, and/or a di- or            oligosaccharide having an N-acetylglucosamine and/or            N-acetylgalactosamine at its non-reducing end.    -   51. Cell according to any one of preferred embodiment 49 or 50,        wherein the cell is modified with one or more gene expression        modules, characterized in that the expression from any of the        expression modules is either constitutive or is created by a        natural inducer.    -   52. Cell according to any one of preferred embodiment 49 to 51,        wherein the cell comprises multiple copies of the same coding        DNA sequence encoding for one protein.    -   53. The cell of any one of preferred embodiments 49 to 52,        wherein the cell is further capable of synthesizing one or more        nucleotide-sugar donor(s) chosen from the list comprising:        GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal,        UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine        (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc),        UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,        UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,        UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or        UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,        UDP-N-acetylfucosamine (UDP-L-FucNAc or        UDP-2-acetamido-2,6-dideoxy-L-galactose),        UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or        UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid,        UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or        UDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose,        CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac,        CMP-Neu5Ac9N₃, CMP-Neu4,5Ac₂, CMP-Neu5,7Ac₂, CMP-Neu5,9Ac₂,        CMP-Neu5,7(8,9)Ac2, UDP-glucuronate, UDP-galacturonate,        GDP-rhamnose, UDP-xylose.    -   54. The cell of any one of preferred embodiments 49 to 53,        wherein the cell is further capable of expressing one or more        glycosyltransferases selected from the list comprising:        fucosyltransferases, sialyltransferases, galactosyltransferases,        glucosyltransferases, mannosyltransferases,        N-acetylglucosaminyltransferases,        N-acetylgalactosaminyltransferases,        N-acetylmannosaminyltransferases, xylosyltransferases,        glucuronyltransferases, galacturonyltransferases,        glucosaminyltransferases, N-glycolyineurarninyltransferases,        rhamnosyltransferases, N-acetylrhamnosyltransferases,        UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine        transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases        and fucosaminyltransferases,        -   preferably, the fucosyltransferase is chosen from the list            comprising alpha-1,2-fucosyltransferase,            alpha-1,3-fucosyltransferase, alpha-1,4-fucosyltransferase            and alpha-1,6-fucosyltransferase,        -   preferably, the sialyltransferase is chosen from the list            comprising alpha-2,3-sialyltransferase,            alpha-2,6-sialyltransferase and alpha-2,8-sialyltransferase,        -   preferably, the galactosyltransferase is chosen from the            list comprising beta-1,3-galactosyltransferase,            N-acetylglucosamine beta-1,3-galactosyltransferase,            beta-1,4-galactosyltransferase, N-acetylglucosamine            beta-1,4-galactosyltransferase,            alpha-1,3-galactosyltransferase and            alpha-1,4-galactosyltransferase,        -   preferably, the glucosyltransferase is chosen from the list            comprising alpha-glucosyltransferase,            beta-1,2-glucosyltransferase, beta-1,3-glucosyltransferase            and beta-1,4-glucosyltransferase,        -   preferably, the mannosyltransferase is chosen from the list            comprising alpha-1,2-mannosyltransferase,            alpha-1,3-mannosyltransferase and            alpha-1,6-mannosyltransferase,        -   preferably, the N-acetylglucosaminyltransferase is chosen            from the list comprising galactoside            beta-1,3-N-acetylglucosaminyltransferase and            beta-1,6-N-acetylglucosaminyltransferase,        -   preferably, the N-acetylgalactosaminyltransferase is an            alpha-1,3-N-acetylgalactosaminyltransferase,        -   preferably, the cell is modified in the expression or            activity of the further glycosyltransferase.    -   55. The cell of any one of preferred embodiments 49 to 54,        wherein the cell is modified in the expression or activity of an        enzyme selected from the group comprising: glucosamine        6-phosphate N-acetyltransferase, phosphatase,        glycosyltransferase, L-glutamine-D-fructose-6-phosphate        aminotransferase, and UDP-glucose 4-epimerase.    -   56. The cell of any one of preferred embodiments 49 to 55,        wherein the cell is unable to convert        N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate,        and/or unable to convert glucosamine-6-phosphate to        fructose-6-phosphate.    -   57. The cell of any one of preferred embodiments 49 to 56,        wherein the cell is modified for enhanced UDP-galactose        production and wherein the modification is chosen from the group        comprising: knock-out of a 5′-nucleotidase/UDP-sugar hydrolase        encoding gene or knock-out of a galactose-1-phosphate        uridylyltransferase encoding gene.    -   58. The cell of any one of preferred embodiments 49 to 57,        wherein the cell is using one or more precursor(s) for the        production of the galactosylated disaccharide or        oligosaccharide, the precursor(s) being fed to the cell from the        cultivation medium.    -   59. The cell of any one of preferred embodiments 49 to 58,        wherein the cell is producing one or more precursor(s) for the        production of the galactosylated disaccharide or        oligosaccharide.    -   60. The cell of any one of preferred embodiments 58 or 59,        wherein the precursor for the production of the galactosylated        disaccharide or oligosaccharide is completely converted into the        galactosylated disaccharide or oligosaccharide.    -   61. The cell of any one of preferred embodiments 49 to 60,        wherein the cell produces the galactosylated disaccharide or        oligosaccharide intracellularly and wherein a fraction or        substantially all of the produced galactosylated disaccharide or        oligosaccharide remains intracellularly and/or is excreted        outside the cell via passive or active transport.    -   62. The cell of any one of preferred embodiments 49 to 61,        wherein the cell expresses a membrane transporter protein or a        polypeptide having transport activity hereby transporting        compounds across the outer membrane of the cell wall,        preferably, the cell is modified in the expression or activity        of the membrane transporter protein or polypeptide having        transport activity.    -   63. The cell of preferred embodiment 62, wherein the membrane        transporter protein or polypeptide having transport activity is        chosen from the list comprising porters,        P—P-bond-hydrolysis-driven transporters, β-barrel porins,        auxiliary transport proteins, putative transport proteins and        phosphotransfer-driven group translocators, preferably, the        porters comprise MFS transporters, sugar efflux transporters and        siderophore exporters, and preferably, the        P—P-bond-hydrolysis-driven transporters comprise ABC        transporters and siderophore exporters.    -   64. The cell of any one of preferred embodiments 62 or 63,        wherein the membrane transporter protein or polypeptide having        transport activity controls the flow over the outer membrane of        the cell wall of the galactosylated disaccharide or        oligosaccharide and/or of one or more precursor(s) and/or        acceptor(s) to be used in the production of the galactosylated        disaccharide or oligosaccharide.    -   65. The cell of any one of preferred embodiments 62 to 64,        wherein the membrane transporter protein or polypeptide having        transport activity provides improved production and/or enabled        and/or enhanced efflux of the galactosylated disaccharide or        oligosaccharide.    -   66. The cell of any one of preferred embodiments 49 to 65,        wherein the cell comprises a modification for reduced production        of acetate compared to a non-modified progenitor.    -   67. The cell of preferred embodiment 66, wherein the cell        comprises a lower or reduced expression and/or abolished,        impaired, reduced or delayed activity of any one or more of the        proteins comprising beta-galactosidase, galactoside        0-acetyltransferase, N-acetylglucosamine-6-phosphate        deacetylase, glucosamine-6-phosphate deaminase,        N-acetylglucosamine repressor, ribonucleotide monophosphatase,        EIICBA-Nag, UDP-glucose:undecaprenyl-phosphate        glucose-1-phosphate transferase, L-fuculokinase, L-fucose        isomerase, N-acetylneuraminate lyase, N-acetylmannosamine        kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man,        EIIC-Man, EIID-Man, ushA, galactose-1-phosphate        uridylyltransferase, glucose-1-phosphate adenylyltransferase,        glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase        isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2,        glucose-6-phosphate isomerase, aerobic respiration control        protein, transcriptional repressor IclR, lon protease,        glucose-specific translocating phosphotransferase enzyme IBC        component ptsG, glucose-specific translocating        phosphotransferase (PTS) enzyme IIBC component malX, enzyme        IIAGlc, beta-glucoside specific PT'S enzyme ii,        fructose-specific PTS multiphosphoryl transfer protein FruA and        FruB, ethanol dehydrogenase aldehyde dehydrogenase,        pyruvate-formate lyase, acetate kinase, phosphoacyltransferase,        phosphate acetyltransferase, pyruvate decarboxylase compared to        a non-modified progenitor.    -   68. The cell of any one of preferred embodiments 49 to 67,        wherein the cell is capable of producing phosphoenolpyruvate        (PEP).    -   69. The cell of any one of preferred embodiments 49 to 68,        wherein the cell is modified for enhanced production and/or        supply of phosphoenolpyruvate (PEP) compared to a non-modified        progenitor.    -   70. The cell of any one of preferred embodiments 49 to 69,        wherein the cell comprises a catabolic pathway for selected        mono-, di- or oligosaccharides that is at least partially        inactivated, the mono-, di-, or oligosaccharides being involved        in and/or required for the production of the galactosylated        disaccharide or oligosaccharide.    -   71. The cell of any one of preferred embodiments 49 to 70,        wherein the cell resists the phenomenon of lactose killing when        grown in an environment in which lactose is combined with one or        more other carbon source(s).    -   72. The cell of any one of preferred embodiments 49 to 71,        wherein the cell is capable of catabolizing a carbon source        selected from the list comprising glucose, fructose, mannose,        galactose, lactose, sucrose, maltose, malto-oligosaccharides,        trehalose, starch, cellulose, hemi-cellulose, molasses,        corn-steep liquor, high-fructose syrup, glycerol, acetate,        citrate, lactate and pyruvate.    -   73. The cell of any one of preferred embodiments 49 to 72 or        method according to any one of preferred embodiments 6 to 48,        wherein the cell is a bacterium, fungus, yeast, a plant cell, an        animal cell, or a protozoan cell,        -   preferably, the bacterium is an Escherichia coli strain,            more preferably an Escherichia coli strain, which is a K-12            strain, even more preferably the Escherichia coli K-12            strain is E. coli MG1655,        -   preferably, the fungus belongs to a genus chosen from the            group comprising Rhizopus, Dictyostelium, Penicillium, Mucor            or Aspergillus,        -   preferably, the yeast belongs to a genus chosen from the            group comprising Saccharomyces, Zygosaccharomyces, Pichia,            Komagataella, Hansenula, Yarrowia, Starmerella,            Kluyveromyces or Debaromyces,        -   preferably, the plant cell is an algal cell or is derived            from tobacco, alfalfa, rice, tomato, cotton, rapeseed, soy,            maize, or corn plant,        -   preferably, the animal cell is derived from non-human            mammals, birds, fish, invertebrates, reptiles, amphibians or            insects or is a genetically modified cell line derived from            human cells excluding embryonic stem cells, more preferably            the human and non-human mammalian cell is an epithelial            cell, an embryonic kidney cell, a fibroblast cell, a COS            cell, a Chinese hamster ovary (CHO) cell, a murine myeloma            cell, an NIH-3T3 cell, a non-mammary adult stem cell or            derivatives thereof, more preferably the insect cell is            derived from Spodoptera frugiperda, Bombyx mori, Mamestra            brassicae, Trichoplusia ni or Drosophila melanogaster,        -   preferably, the protozoan cell is a Leishmania tarentolae            cell.    -   74. The cell of preferred embodiment 73 or method according to        preferred embodiment 73, wherein the cell is a viable        Gram-negative bacterium that comprises a reduced or abolished        synthesis of poly-N-acetyl-glucosamine (PNAG), Enterobacterial        Common Antigen (ECA), cellulose, colanic acid, core        oligosaccharides, Osmoregulated Periplasmic Glucans (OPG),        Glucosylglycerol, glycan, and/or trehalose compared to a        non-modified progenitor.    -   75. The cell of any one of preferred embodiments 49 to 74,        wherein the cell produces a mixture of charged, preferably        sialylated, and/or neutral di- and/or oligosaccharides        comprising at least one galactosylated disaccharide or        oligosaccharide.    -   76. The cell of any one of preferred embodiments 49 to 74,        wherein the cell produces a mixture of charged, preferably        sialylated, and/or neutral oligosaccharides comprising at least        one galactosylated oligosaccharide.    -   77. Use of the cell of any one of preferred embodiments 49 to        74, or a method according to any one of preferred embodiments 1        to 41, 73 to 74 for the production of a galactosylated        disaccharide or oligosaccharide.    -   78. Use of the cell of any one of preferred embodiments 49 to        75, or a method according to any one of preferred embodiments 1        to 42, 73 to 74 for the production of a mixture of charged,        preferably sialylated, and/or neutral di- and/or        oligosaccharides comprising at least one galactosylated        disaccharide or oligosaccharide.    -   79. Use of the cell of any one of preferred embodiments 49 to        76, or a method according to any one of preferred embodiments 1        to 43, 73 to 74 for the production of a mixture of charged,        preferably sialylated, and/or neutral oligosaccharides        comprising at least one galactosylated oligosaccharide.

The following examples will serve as further illustration andclarification of the disclosure and are not intended to be limiting.

DETAILED DESCRIPTION Examples Example 1: Materials and MethodsEscherichia coli

Media: The Luria Broth (LB) medium consisted of 1% tryptone peptone(Difco, Erembodegem, BE), 0.5% yeast extract (Difco) and 0.5% sodiumchloride (VWR. Leuven, BE). The medium used in the cultivationexperiments in 96-well plates or in shake flasks contained 2.00 g/LNH4Cl, 5.00 g/L (NH4)2SO4, 2.993 g/L KH2PO4, 7.315 g/L K2HPO4, 8.372 g/LMOPS, 0.5 g/L NaCl, 0.5 g/L MgSO4.7H2O, 30 g/L sucrose or 30 g/Lglycerol, 1 ml/L vitamin solution, 100 μl/L molybdate solution, and 1mL/L selenium solution. As specified in the respective examples, 0.30g/L sialic acid, 20 g/L lactose, 20 g/L LacNAc and/or 20 g/L LNB wereadditionally added to the medium as precursor(s). The medium was set toa pH of 7 with 1 M KOH. Vitamin solution consisted of 3.6 g/LFeCl2.4H2O, 5 g/L CaCl2·2H2O, 1.3 g/L MnCl2·2H2O, 0.38 g/L CuCl2·2H2O,0.5 g/L CoCl2·6H2O, 0.94 g/L ZnCl2, 0.0311 g/L H3BO4, 0.4 g/LNa2EDTA·2H2O and 1.01 g/L thiamine·HCl. The molybdate solution contained0.967 g/L NaMoO4·2H2O. The selenium solution contained 42 g/L Seo2.

The minimal medium for fermentations contained 6.75 g/L NH4Cl, 1.25 g/L(NH4)2SO4, 2.93 g/L KH2PO4 and 7.31 g/L KH2PO4, 0.5 g/L NaCl, 0.5 g/LMgSO4.7H2O, 30 g/L sucrose or 30 g/L glycerol, 1 mL/L vitamin solution,100 μL/L molybdate solution, and 1 mL/L selenium solution with the samecomposition as described above. As specified in the respective examples,0.30 g/L sialic acid and/or 20 g/L lactose, were additionally added tothe minimal medium for fermentations as precursor(s).

Complex medium was sterilized by autoclaving (121° C., 21 min.) andminimal medium by filtration (0.22 μm Sartorius). When necessary, themedium was made selective by adding an antibiotic: e.g., chloramphenicol(20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/orkanamycin (50 mg/L).

Plasmids: pKD46 (Red helper plasmid, Ampicillin resistance), pKD3(contains an FRT-flanked chloramphenicol resistance (cat) gene), pKD4(contains an FRT-flanked kanamycin resistance (kan) gene), and pCP20(expresses FLP recombinase activity) plasmids were obtained from Prof R.Cunin (Vrije Universiteit Brussel, BE in 2007).

Plasmids were maintained in the host E. coli DH5alpha (F⁻,phi80dlacZΔM15, Δ(lacZYA-argF) U169, deoR, recAl, endAl, hsdR17(rk⁻,mk⁺), phoA, supE44, lambda⁻, thi-1, gyrA96, relAl) bought fromInvitrogen.

Strains and Mutaions: Escherichia coli K12 MG1655 [λ⁻, F⁻, rph-1] wasobtained from the Coli Genetic Stock Center (US), CGSC Strain #: 7740,in March 2007. Gene disruptions, gene introductions and genereplacements were performed using the technique published by Datsenkoand Wanner (PNAS 97 (2000), 6640-6645). This technique is based onantibiotic selection after homologous recombination performed by lambdaRed recombinase. Subsequent catalysis of a flippase recombinase ensuresremoval of the antibiotic selection cassette in the final productionstrain.

Transformants carrying a Red helper plasmid pKD46 were grown in 10 mL LBmedia with ampicillin, (100 mg/L) and L-arabinose (10 mM) at 30° C. toan OD₆₀₀ nm of 0.6. The cells were made electrocompetent by washing themwith 50 mL of ice-cold water, a first time, and with 1 mL ice coldwater, a second time. Then, the cells were resuspended in 50 μL ofice-cold water. Electroporation was done with 50 μL of cells and 10-100ng of linear double-stranded-DNA product by using a Gene Pulser™(BioRad) (600 Ω, 25 μFD, and 250 volts).

After electroporation, cells were added to 1 mL LB media incubated 1hour at 37° C., and finally spread onto LB-agar containing 25 mg/L ofchloramphenicol or 50 mg/L of kanamycin to select antibiotic-resistanttransformants. The selected mutants were verified by PCR with primersupstream and downstream of the modified region and were grown in LB-agarat 42° C. for the loss of the helper plasmid. The mutants were testedfor ampicillin sensitivity.

The linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 andtheir derivates as template. The primers used had a part of the sequencecomplementary to the template and another part complementary to the sideon the chromosomal DNA where the recombination must take place. For thegenomic knock-out, the region of homology was designed 50-nt upstreamand 50-nt downstream of the start and stop codon of the gene ofinterest. For the genomic knock-in, the transcriptional starting point(+1) had to be respected. PCR products were PCR-purified, digested withDpnl, re-purified from an agarose gel, and suspended in elution buffer(5 mM Tris, pH 8.0).

Selected mutants were transformed with pCP20 plasmid, which is anampicillin- and chloramphenicol-resistant plasmid that showstemperature-sensitive replication and thermal induction of FLPsynthesis. The ampicillin-resistant transformants were selected at 30°C., after which a few were colony purified in LB at 42° C. and thentested for loss of all antibiotic resistance and of the FLP helperplasmid. The gene knock-outs and knock-ins are checked with controlprimers.

In one example for GDP-fucose and fucosylated oligosaccharideproduction, the mutant strain was derived from E. coli K12 MG1655comprising knock-outs of the E. coli wcaJ and thyA genes and genomicknock-ins of constitutive expression constructs containing a sucrosetransporter like, e.g., CscB originating from E. coli W (UniProt IDEOIXR1), a fructose kinase like, e.g., frk originating from Zymomonasmobilis (ZmFrk) (UniProt ID Q03417), a sucrose phosphorylase like, e.g.,BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6),additionally comprising expression plasmids with constitutive expressionconstructs for an alpha-1,2-fucosyltransferase like, e.g., HpFutC fromH. pylori (GenBank No. AAD29863.1) and/or analpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProtID 030511) and with a constitutive expression construct for the E. colithyA (UniProt ID P0A884) as selective marker. The constitutiveexpression constructs of the fucosyltransferase genes can also bepresent in the mutant E. coli strain via genomic knock-ins. GDP-fucoseproduction can further be optimized in the mutant E. coli strain bygenomic knock-outs of the E. coli genes comprising glgC, agp, pfkA,pfkB, pgi, arcA, iclR, pgi and lon as described in WO2016075243 andWO2012007481. GDP-fucose production can additionally be optimizedcomprising genomic knock-ins of constitutive expression constructs for amannose-6-phosphate isomerases like, e.g., manA from E. coli (UniProt IDP00946), a phosphomannomutase like, e.g., manB from E. coli (UniProt IDP24175), a mannose-1-phosphate guanylyltransferase like, e.g., manC fromE. coli (UniProt ID P24174), a GDP-mannose 4,6-dehydratase like, e.g.,gmd from E. coli (UniProt ID P0AC88) and a GDP-L-fucose synthase like,e.g., fcl from E. coli (UniProt ID P32055). GDP-fucose production canalso be obtained by genomic knock-outs of the E. coli fucK and fucIgenes together with genomic knock-ins of constitutive expressionconstructs containing fucose permease like, e.g., fucP from E. coli(UniProt ID P11551) and a bifunctional enzyme with fucosekinase/fucose-1-phosphate guanylyltransferase activity like, e.g., fkpfrom Bacteroides fragilis (UniProt ID SUV40286.1). If the mutant strainproducing GDP-fucose was intended to make fucosylated lactosestructures, the strain was additionally modified with genomic knock-outsof the E. coli LacZ, LacY and LacA genes and with a genomic knock-in ofa constitutive expression construct for a lactose permease like, e.g.,the E. coli LacY (UniProt ID P02920).

Alternatively, and/or additionally, GDP-fucose and/or fucosylatedoligosaccharide production can further be optimized in the mutant E.coli strains with genomic knock-ins of constitutive transcriptionalunits comprising a membrane transporter protein like, e.g., MdfA fromCronobacter muytjensii (UniProt ID AOA2T7ANQ9), MdfA from Citrobacteryoungae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID POAEY8), MdfAfrom Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli(UniProt ID A0A024L207) or iceT from Citrobacter youngae (UniProt IDD4B8A6).

In one example for sialic acid production, the mutant strain was derivedfrom E. coli K12 MG1655 comprising genomic knock-ins of constitutivetranscriptional units containing one or more copies of a glucosamine6-phosphate N-acetyltransferase like, e.g., GNA1 from Saccharomycescerevisiae (UniProt ID P43577), an N-acetylglucosamine 2-epimerase like,e.g., AGE from Bacteroides ovatus (UniProt ID A7LVG6) and anN-acetylneuraminate synthase like, e.g., from Neisseria meningitidis(UniProt ID E0NCD4) or Campylobacter jejuni (UniProt ID Q93MP9).

Alternatively, and/or additionally, sialic acid production can beobtained by genomic knock-ins of constitutive transcriptional unitscontaining a UDP-N-acetylglucosamine 2-epimerase like, e.g., NeuC fromC. jejuni (UniProt ID Q93MP8) and an N-acetylneuraminate synthase like,e.g., from Neisseria meningitidis (UniProt ID E0NCD4) or Campylobacterjejuni (UniProt ID Q93MP9).

Alternatively and/or additionally, sialic acid production can beobtained by genomic knock-ins of constitutive transcriptional unitscontaining a phosphoglucosamine mutase like, e.g., glmM from E. coli(UniProt ID P31120), an N-acetylglucosamine-1-phosphateuridyltransferase/glucosamine-1-phosphate acetyltransferase like, e.g.,glmU from E. coli (UniProt ID P0ACC7), a UDP-N-acetylglucosamine2-epimerase like, e.g., NeuC from C. jejuni (UniProt ID Q93MP8) and anN-acetylneuraminate synthase like, e.g., from Neisseria meningitidis(UniProt ID E0NCD4) or Campylobacter jejuni (UniProt ID Q93MP9).

Alternatively, and/or additionally, sialic acid production can beobtained by genomic knock-ins of constitutive transcriptional unitscontaining a bifunctional UDP-GlcNAc 2-epimerase/N-acetylmannosaminekinase like, e.g., from Mus musculus (strain C57BL/6J) (UniProt IDQ91WG8), an N-acylneuraminate-9-phosphate synthetase like, e.g., fromPseudomonas sp. UW4 (UniProt ID K9NPH9) and anN-acylneuraminate-9-phosphatase like, e.g., from Candidatus Magnetomorumsp. HK-1 (UniProt ID KPA15328.1) or from Bacteroides thetaiotaomicron(UniProt ID Q8A712).

Alternatively, and/or additionally, sialic acid production can beobtained by genomic knock-ins of constitutive transcriptional unitscontaining a phosphoglucosamine mutase like, e.g., glmM from E. coli(UniProt ID P31120), an N-acetylglucosamine-1-phosphateuridyltransferase/glucosamine-1-phosphate acetyltransferase like, e.g.,glmU from E. coli (UniProt ID P0ACC7), a bifunctional UDP-GlcNAc2-epimerase/N-acetylmannosamine kinase like, e.g., from M. musculus(strain C57BL/6J) (UniProt ID Q91WG8), an N-acylneuraminate-9-phosphatesynthetase like, e.g., from Pseudomonas sp. UW4 (UniProt ID K9NPH9) andan N-acylneuraminate-9-phosphatase like, e.g., from CandidatusMagnetomorum sp. HK-1 (UniProt ID KPA15328.1) or from Bacteroidesthetaiotaomicron (UniProt ID Q8A712).

Sialic acid production can further be optimized in the mutant E. colistrain with genomic knock-outs of the E. coli genes comprising any oneor more of nagA, nagB, nagC, nagD, nagE, nanA, nanE, nanK, manX, manYand manZ as described in WO 2018122225, and/or genomic knock-outs of theE. coli genes comprising any one or more of nanT, poxB, idhA, adhE,aldB, pflA, pflC, ybiY, ackA and/or pta and with genomic knock-ins ofconstitutive transcriptional units comprising one or more copies of anL-glutamine-D-fructose-6-phosphate aminotransferase like, e.g., themutant glmS*54 from E. coli (differing from the wild-type E. coli glmS,having UniProt ID P17169, by an A39T, an R250C and a G472S mutation asdescribed by Deng et a1. (Biochimie 88, 419-29 (2006)), preferably onephosphatase like any one or more of, e.g., the E. coli genes comprisingaphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX,YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL,YjjG, YrfG and YbiU or PsMupP from Pseudomonas putida, ScDOG1 from S.cerevisiae or BsAraL from Bacillus subtilis as described in WO2018122225 and an acetyl-CoA synthetase like, e.g., acs from E. coli(UniProt ID P27550).

For sialylated oligosaccharide production, the sialic acid productionstrains were further modified to express an N-acylneuraminatecytidylyltransferase like, e.g., the NeuA enzyme from C. jejuni (UniProtID Q93MP7), the NeuA enzyme from Haemophilus influenzae (GenBank No.AGV11798.1) or the NeuA enzyme from Pasteurella multocida (GenBank No.AMK07891.1) and to express one or more copies of a beta-galactosidealpha-2,3-sialyltransferase like, e.g., PmultST3 from P. multocida(UniProt ID Q9CLP3) or a PmultST3-like polypeptide consisting of aminoacid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactosidealpha-2,3-sialyltransferase activity, NmeniST3 from N. meningitidis(GenBank No. ARC07984.1) or PmultST2 from P. multocida subsp. multocidastr. Pm70 (GenBank No. AAK02592.1), a beta-galactosidealpha-2,6-sialyltransferase like, e.g., PdST6 from Photobacteriumdamselae (UniProt ID O66375) or a PdST6-like polypeptide consisting ofamino acid residues 108 to 497 of UniProt ID O66375 havingbeta-galactoside alpha-2,6-sialyltransferase activity orP-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1)or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues18 to 514 of UniProt ID A8QYL1 having beta-galactosidealpha-2,6-sialyltransferase activity, and/or analpha-2,8-sialyltransferase like, e.g., from M. musculus (UniProt IDQ64689). Constitutive transcriptional units of the N-acylneuraminatecytidylyltransferase and the sialyltransferases can be delivered to themutant strain either via genomic knock-in or via expression plasmids. Ifthe mutant strains producing sialic acid and CMP-sialic acid wereintended to make sialylated lactose structures, the strains wereadditionally modified with genomic knock-outs of the E. coli LacZ, LacYand LacA genes and with a genomic knock-in of a constitutivetranscriptional unit for a lactose permease like, e.g., E. coli LacY(UniProt ID P02920). All mutant strains producing sialic acid,CMP-sialic acid and/or sialylated oligosaccharides could optionally beadapted for growth on sucrose via genomic knock-ins of constitutivetranscriptional units containing a sucrose transporter like, e.g., CscBfrom E. coli W (UniProt ID E0IXR1), a fructose kinase like, e.g., Frkoriginating from Z. mobilis (UniProt ID Q03417) and a sucrosephosphorylase like, e.g., BaSP from B. adolescentis (UniProt ID A0ZZH6).

Alternatively, and/or additionally, sialic acid and/or sialylatedoligosaccharide production can further be optimized in the mutant E.coli strains with genomic knock-ins of constitutive transcriptionalunits comprising a membrane transporter protein like, e.g., a sialicacid transporter like, e.g., nanT from E. coli K-12 MG1655 (UniProt IDP41036), nanT from E. coli O6:H1 (UniProt ID Q8FD59), nanT from E. coliO157:H7 (UniProt ID Q8X9G8) or nanT from E. albertii (UniProt ID B1EFH1)or a porter like, e.g., EntS from E. coli (UniProt ID P24077), EntS fromKluyvera ascorbata (UniProt ID A0A378GQ13), EntS from Salmonellaenterica subsp. arizonae (UniProt ID A0A6Y2K4E8), MdfA from Cronobactermuytjensii (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter youngae(UniProt ID D4BC23), MdfA from E. coli (UniProt ID POAEY8), MdfA fromYokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProtID A0A024L207), iceT from Citrobacter youngae (UniProt ID D4B8A6), SetAfrom E. coli (UniProt ID P31675), SetB from E. coli (UniProt ID P33026)or SetC from E. coli (UniProt ID P31436) or an ABC transporter like,e.g., oppF from E. coli (UniProt ID P77737), lmrA from Lactococcuslactis subsp. lactis bv. diacetylactis (UniProt ID A0A1V0NEL4), orBlon_2475 from Bifidobacterium longum subsp. infantis (UniProt IDB7GPD4).

In an example for enhanced UDP-galactose production, the E. coli K12MG1655 strain was modified with genomic knock-outs of any one or more ofthe E. coli ushA, galT, ldhA and agp genes and with a genomic knock-inof a constitutive expression construct for the UDP-glucose4-epimerase(galE) of E. coli (UniProt ID P09147).

In an example for enhanced UDP-GlcNAc production, the E. coli K12 MG1655strain was modified with a genomic knock-in of a constitutivetranscriptional unit for an L-glutamine D-fructose-6-phosphateaminotransferase like, e.g., the mutant glmS*54 from E. coli (differingfrom the wild-type E. coli glmS protein, having UniProt ID P17169, by anA39T, an R250C and a G472S mutation as described by Deng et a1.(Biochimie 2006, 88: 419-429).

In an example to produce lacto-N-triose (LN3, GlcNAc-b1,3-Gal-b1,4-Glc),the mutant strain was derived from E. coli K12 MG1655 and modified witha knock-out of the E. coli lacZ, lacY, lacA and nagB genes and withgenomic knock-ins of constitutive transcriptional units for a lactosepermease like, e.g., the E. coli LacY (UniProt ID P02920) and agalactoside beta-1,3-N-acetylglucosaminyltransferase like, e.g., lgtA(UniProt ID Q9JXQ6) from N. meningitidis.

In an example for production of LN3 derived oligosaccharides likelacto-N-tetraose (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), the mutantLN3 producing strain was further modified with a constitutivetranscriptional unit delivered to the strain either via genomic knock-inor from an expression plasmid for an N-acetylglucosaminebeta-1,3-galactosyltransferase chosen from the list comprising SEQ IDNOs: 3, 4, 6, 7, 8 and 9.

In an example for production of LN3 derived oligosaccharides likelacto-N-neotetraose (LNnT, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), themutant LN3 producing strain was further modified with a constitutivetranscriptional unit delivered to the strain either via genomic knock-inor from an expression plasmid for an N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34,37, 38 and 39.

In an example for production of lacto-N-biose (LNB, Gal-b1,3-GlcNAc) andLNB-derived oligosaccharides the strains were modified with genomicknock-ins or expression plasmids comprising constitutive transcriptionalunits for one or more copies of a glucosamine 6-phosphateN-acetyltransferase like, e.g., GNA1 from S. cerevisiae (UniProt IDP43577) and an N-acetylglucosamine beta-1,3-galactosyltransferase chosenfrom the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9.

In an example for production of N-acetyllactosamine (LacNAc,Gal-b1,4-GlcNAc) and LacNAc-derived oligosaccharides the strains weremodified with genomic knock-ins or expression plasmids comprisingconstitutive transcriptional units for one or more copies of aglucosamine 6-phosphate N-acetyltransferase like, e.g., GNA1 from S.cerevisiae (UniProt ID P43577) and an N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34,37, 38 and 39.

The mutant LNB, LacNAc, LN3, LNT and LNnT producing E. coli strains canalso optionally be adapted for growth on sucrose via genomic knock-insof constitutive transcriptional units containing a sucrose transporterlike, e.g., CscB from E. coli W (UniProt ID EOIXR1), a fructose kinaselike, e.g., Frk originating from Zymomonas mobilis (UniProt ID Q03417)and a sucrose phosphorylase like, e.g., BaSP originating fromBifidobacterium adolescentis (UniProt ID A0ZZH6).

Alternatively, and/or additionally, production of LN3, LNT, LNnT, LNB,LacNAc and oligosaccharides derived thereof can further be optimized inthe mutant E. coli strains with genomic knock-ins of a constitutivetranscriptional unit comprising a membrane transporter protein like,e.g., MdfA from Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), MdfAfrom Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProtID POAEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceTfrom E. coli (UniProt ID A0A024L207) or iceT from Citrobacter youngae(UniProt ID D4B8A6).

Preferably, but not necessarily, any one or more of theglycosyltransferases, the proteins involved in nucleotide-activatedsugar synthesis and/or membrane transporter protein were N- and/orC-terminally fused to a solubility enhancer tag like, e.g., a SUMO-tag,an MBP-tag, His, FLAG, Strep-II, Halo-tag, NusA, thioredoxin, GST and/orthe Fh8-tag to enhance their solubility (Costa et al., Front. Microbiol.2014, https://doi.org/10.3389/fmicb.2014.00063; Fox et al., Protein Sci.2001, 10(3), 622-630; Jia and Jeaon, Open Biol. 2016, 6: 160196).

Optionally, the mutant E. coli strains were modified with a genomicknock-in of a constitutive transcriptional unit encoding a chaperoneprotein like, e.g., DnaK, DnaJ, GrpE or the GroEL/ES chaperonin system(Baneyx F., Palumbo J. L. (2003) Improving Heterologous Protein Foldingvia Molecular Chaperone and Foldase Co-Expression. In: Vaillancourt P.E. (eds) E. coli Gene Expression Protocols. Methods in MolecularBiology™, vol 205. Humana Press).

Optionally, the mutant E. coli strains are modified to create aglycominimized E. coli strain comprising genomic knock-out of any one ormore of non-essential glycosyltransferase genes comprising pgaC, pgaD,rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, wcaI, wcaJ, wcaL,waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ, wbbl,arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsA andyaiP.

All constitutive promoters and UTRs originated from the librariesdescribed by De Mey et a1. (BMC Biotechnology, 2007), Dunn et a1.(Nucleic Acids Res. 1980, 8(10), 2119-2132), Kim and Lee (FEBS letters1997, 407(3), 353-356) and Mutalik et a1. (Nat. Methods 2013, No. 10,354-360).

The SEQ ID NOs described in the disclosure are summarized in Table 1.

All genes were ordered synthetically at Twist Bioscience(twistbioscience.com) or IDT (eu.idtdna.com) and the codon usage wasadapted using the tools of the supplier.

All strains are stored in cryovials at −80° C. (overnight LB culturemixed in a 1:1 ratio with 70% glycerol).

TABLE 1 Overview of SEQ ID NOs described in the disclosure SEQ Countryof origin ID of digital NO Organism Origin sequence information 01 N.A.Synthetic Artificial sequence 02 N.A. Synthetic Artificial sequence 03Corynebacterium glutamicum Synthetic Japan 04 Photobacterium leiognathiSynthetic USA 05 N.A. Synthetic Artificial sequence 06 A. thalianaSynthetic USA 07 Trypanosoma brucei Synthetic Scotland 08 Mus musculusSynthetic USA 09 Homo sapiens Synthetic Unknown 10 N.A. SyntheticArtificial sequence 11 N.A. Synthetic Artificial sequence 12 N.A.Synthetic Artificial sequence 13 N.A. Synthetic Artificial sequence 14N.A. Synthetic Artificial sequence 15 Basilea psittacipulmonis SyntheticSwitzerland 16 Neisseria arctica Synthetic USA 17 Glaesserella parasuisSynthetic USA 18 Actinobacillus seminis Synthetic Australia 19 Alysiellafiliformis Synthetic Australia 20 Conchiformibius steedae SyntheticUnited Kingdom 21 Acinetobacter haemolyticus Synthetic South-Korea 22Campylobacter pylori Synthetic Switzerland 23 Histophilus somniSynthetic Canada 24 N.A. Synthetic Artificial sequence 25 N.A. SyntheticArtificial sequence 26 Streptococcus pneumoniae Synthetic USA 27 Hafniaalvei Synthetic Unknown 28 N.A. Synthetic Artificial sequence 29Mycolicibacterium flavescens Synthetic USA 30 Sphingomonas sp. SyntheticUnited Kingdom 31 Parachlamydiaceae bacterium Synthetic Japan HS-T3 32Coxiella sp. DG_40 Synthetic USA 33 Corallococcus exercitus SyntheticUnited Kingdom 34 Hypericibacter adhaerens Synthetic Germany 35 N.A.Synthetic Artificial sequence 36 N.A. Synthetic Artificial sequence 37Bacteroides vulgatus Synthetic United Kingdom 38 Prevotella copriSynthetic USA 39 Pseudomonas fluorescens Synthetic USA 90F12-2

Cultivation Conditions: A pre-culture of 96-well microtiter plateexperiments was started from a cryovial, in 150 μL LB and was incubatedovernight at 37° C. on an orbital shaker at 800 rpm. This culture wasused as inoculum for a 96-well square microtiter plate, with 400 μL MMsfmedium by diluting 400×. These final 96-well culture plates were thenincubated at 37° C. on an orbital shaker at 800 rpm for 72 hours, orshorter, or longer. To measure sugar concentrations at the end of thecultivation experiment whole broth samples were taken from each well byboiling the culture broth for 15 min. at 60° C. before spinning down thecells (=average of intra- and extracellular sugar concentrations).

A pre-culture for the bioreactor was started from an entire 1 mLcryovial of a certain strain, inoculated in 250 mL or 500 mL of MMsfmedium in a 1 L or 2.5 L shake flask and incubated for 24 hours at 37°C. on an orbital shaker at 200 rpm. A 5 L bioreactor was then inoculated(250 mL inoculum in 2 L batch medium); the process was controlled byMFCS control software (Sartorius Stedim Biotech, Melsungen, Germany).Culturing conditions were set to 37° C., and maximal stirring; pressuregas flow rates were dependent on the strain and bioreactor. The pH wascontrolled at 6.8 using 0.5 M H2SO4 and 20% NH40H. The exhaust gas wascooled. 10% solution of silicone antifoaming agent was added whenfoaming raised during the fermentation.

Optical Density: Cell density of the cultures was frequently monitoredby measuring optical density at 600 nm (Implen Nanophotometer NP80,Westburg, BE or with a Spark 10 M microplate reader, Tecan, CH).

Analytical Analysis: Standards such as, but not limited to, sucrose,glucose, N-acetylglucosamine, N-acetyllactosamine, lacto-N-biose,fucosylated N-acetyllactosamine (2′FLAcNAc, 3-FlacNAc), fucosylatedlacto-N-biose (2′FLNB, 4-FLNB), sialylated N-acetyllactosamine(3′SLacNAc, 6′SLacNAc were purchased from Carbosynth (UK), Elicityl(France) and IsoSep (Sweden). Other compounds were analyzed within-house made standards.

N-acetylglucosamine and N-acetyllactosamine were analyzed on a DionexHPAEC system with pulsed amperometric detection (PAD). A volume of 5 μLof sample was injected on a Dionex CarboPac PA1 column 4×250 mm with aDionex CarboPac PA1 guard column 4×50 mm. The column temperature was 20°C. The separation was performed using 3 eluents: A) deionized water B)200 mM Sodium hydroxide and C) 500 mM Sodium acetate. The elutionprofile was applied as follow: 0-10 min. 50% A and 50% B; 10-18 min.50-44% A and 50% B; 18-28 min. 44% A and 50% B; 28-32 min. 44-30.8% Aand 50% B; 32-39 min. 30.8% A and 50% B; 39-40 min. 30.8-2% A and 50% B;40-43 min. 2% A and 50% B; 43-44 min. 2-50% A and 50% B; 44-50 min. 50%A and 50% B. Flow rate was 1.0 mL/minute.

N-acetylglucosamine, N-acetyllactosamine, lacto-N-biose, fucosylatedN-acetyllactosamine and fucosylated LNB were analyzed on a WatersAcquity H-class UPLC with Evaporative Light Scattering Detector (ELSD)or a Refractive Index (RI) detection. A volume of 0.7 μL sample wasinjected on a Waters Acquity UPLC BEH Amide column (2.1×100 mm; 130 Å;1.7 μm) column with an Acquity UPLC BEH Amide VanGuard column, 130 Å,2.1×5 mm. The column temperature was 50° C. The mobile phase consists ofa 1/4 water and 3/4 acetonitrile solution were 0.2% Triethylamine wasadded. The method was isocratic with a flow of 0.130 mL/minute. The ELSdetector had a drift tube temperature of 50° C. and N₂ gas pressure was50 psi, gain 200 and data rate is 10 pps. The temperature of the RIdetector was set at 35° C.

Sialylated N-acetyllactosamine and sialylated lacto-N-biose wereanalyzed with a Waters Acquity H-Class UPLC with Refractive Index (RI)detection. A volume of 0.5 μL sample was injected on a Waters AcquityUPLC BEH Amide column (2.1×100 mm with 1.7 μm particle size) with amobile phase containing 70 mL acetonitrile, 26 mL 150 mM ammoniumacetate in ultrapure water and 4 mL methanol with 0.05% pyrrolidineadded. The method was isocratic with a flow rate of 0.150 mL/minute. Thetemperature of the column was set at 50° C.

Sugars at low concentrations (below 50 mg/L) were analyzed on a DionexHPAEC system with pulsed amperometric detection (PAD). A volume of 5 μLof sample was injected on a Dionex CarboPac PA200 column 4×250 mm with aDionex CarboPac PA200 guard column 4×50 mm. The column temperature wasset to 30° C. A gradient was used wherein eluent A was deionized water,wherein eluent B was 200 mM Sodium hydroxide and wherein eluent C was500 mM Sodium acetate. The oligosaccharides were separated in 60 min.while maintaining a constant ratio of 25% of eluent B using thefollowing gradient: an initial isocratic step maintained for 10 min. of75% of eluent A, an initial increase from 0 to 4% of eluent C over 8min., a second isocratic step maintained for 6 min. of 71% of eluent Aand 4% of eluent C, a second increase from 4 to 12% of eluent C over 2.6min., a third isocratic step maintained for 3.4 min. of 63% of eluent Aand 12% of eluent C and a third increase from 12 to 48% of eluent C over5 min. As a washing step 48% of eluent C was used for 3 min. For columnequilibration, the initial condition of 75% of eluent A and 0% of eluentC was restored in 1 minute and maintained for 11 min. The applied flowwas 0.5 mL/minute.

Normalization of the data: For all types of cultivation conditions, dataobtained from the mutant strains was normalized against data obtained inidentical cultivation conditions with reference strains.

Example 2: Production of GlcNAc in a Modified E. coli Host

In this example, a wild-type E. coli K-12 MG1655 was modified with aknock-out of the homologous E. coli N-acetylglucosamine-6-phosphatedeacetylase (nagA) gene and the E. coli glucosamine-6-phosphatedeaminase (nagB) gene and then transformed with an expression plasmidcomprising a constitutive transcriptional unit for the glucosamine6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae(UniProt ID P43577). The thus obtained mutant E. coli strain producedGlcNAc in whole broth samples when evaluated in a growth experiment,according to the culture conditions in Example 1, in which the culturemedium contained glycerol.

Example 3: Production of GlcNAc in a Modified E. coli Host

The mutant E. coli strain modified to produce GlcNAc as described inExample 2 was further transformed with a second compatible expressionplasmid comprising a constitutive transcriptional unit for the mutatedvariant of the L-glutamine-D-fructose-6-phosphate aminotransferase(glmS*54) from E. coli differing from the wild-type glmS protein(UniProt ID P17169) by an A39T, an R250C and a G472S mutation asdescribed by Deng et a1. (Biochimie 2006: 88, 419-429). The novel strainproduced GlcNAc in whole broth samples when evaluated in a growthexperiment, according to the culture conditions in Example 1, in whichthe culture medium contained glycerol.

Example 4: Production of G/cNAc in a Modified E. coli Host

A wild-type E. coli K-12 MG1655 was modified with a knock-out of the E.coli nagA and the nagB genes and a genomic knock-in of a constitutivetranscriptional unit for GNA1 from S. cerevisiae (UniProt ID P43577).The thus obtained mutant E. coli strain produces GlcNAc in whole brothsamples when evaluated in a growth experiment, according to the cultureconditions in Example 1, in which the culture medium contains glycerol.

Example 5: Production of GlcNAc in a Modified E. coli Host

The mutant E. coli strain modified to produce GlcNAc as described inExample 4 was further transformed with an expression plasmid comprisinga constitutive transcriptional unit for glmS*54 from E. coli differingfrom the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250Cand a G472S mutation as described by Deng et a1. (Biochimie 2006: 88,419-429). The novel strain produced GlcNAc in whole broth samples whenevaluated in a growth experiment, according to the culture conditions inExample 1, in which the culture medium contained glycerol.

Example 6: Production of GlcNAc in a Modified E. coli Host

The mutant E. coli strain modified to produce GlcNAc as described inExample 4 was further transformed with a genomic knock-in comprising aconstitutive transcriptional unit for glmS*54 from E. coli differingfrom the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250Cand a G472S mutation as described by Deng et a1. (Biochimie 2006: 88,419-429). The novel strain produced GlcNAc in whole broth samples whenevaluated in a growth experiment, according to the culture conditions inExample 1, in which the culture medium contained glycerol.

Example 7: Production of G/cNAc in a Modified E. coli Host

The mutant E. coli strain modified to produce GlcNAc as described inExample 2 was further modified with a genomic knock-in comprising aconstitutive transcriptional unit for glmS*54 from E. coli differingfrom the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250Cand a G472S mutation as described by Deng et a1. (Biochimie 2006: 88,419-429). The novel strain produced GlcNAc in whole broth samples whenevaluated in a growth experiment, according to the culture conditions inExample 1, in which the culture medium contained glycerol.

Example 8: Production of LacNAc or LNB in a Modified E. coli Host

The mutant GlcNAc-producing E. coli K-12 MG1655 strains having a nagABKO and expressing GNA1 (UniProt ID P43577) with or without additionalexpression of the mutant glmS*54 differing from the wild-type glmSprotein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation asdescribed by Deng et a1. (Biochimie 2006: 88, 419-429) as described inExamples 2 and 4 to 7 are in a next example transformed with a plasmidcontaining a constitutive transcriptional unit to express either anN-acetylglucosamine β1,4-galactosyltransferase chosen from the listcomprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29,30, 31, 32, 33, 34, 37, 38 and 39 or an N-acetylglucosamineβ1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs:3, 4, 6, 7, 8, and 9.

Each of the novel strains expressing an N-acetylglucosamineβ1,4-galactosyltransferase are evaluated for production of GlcNAc andLacNAc in whole broth samples in a growth experiment according to theculture conditions in Example 1 in which the culture medium containsglycerol.

Each of the novel strains expressing an N-acetylglucosamine β1,3-areevaluated for production of GlcNAc and LNB in whole broth samples in agrowth experiment according to the culture conditions in Example 1 inwhich the culture medium contains glycerol.

Example 9: Production of GlcNAc and LacNAc in Modified E. coli Hosts

An E. coli K-12 MG1655 mutant strain optimized for GDP-fucose productionas described in Example 1, is further modified with a knock-out of theE. coli nagA and nagB genes and with a genomic knock-in of aconstitutive expression construct of an N-acetylglucosamineβ1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37,38 and 39. In a next step, cells of the mutant strain are transformedwith an expression vector comprising constitutive transcriptional unitsof the mutant glmS*54 from E. coli (differing from the wild-type glmSprotein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation)and GNA1 from S. cerevisiae (UniProt ID P43577). The novel strain isevaluated for production of GlcNAc and LacNAc in a growth experimentaccording to the culture conditions provided in Example 1, in which theculture medium contains sucrose. The strain is grown for 72 hours inmultiple wells of a 96-well plate; afterwards the culture broth isharvested and the GlcNAc and LacNAc are analyzed on UPLC.

Example 10: Production of GlcNAc and LacNAc in Modified E. coli Hosts

In a next experiment, an E. coli K-12 MG1655 strain producing sialicacid, as described in Example 1 and containing knock-outs of the E. colinagA and nagB genes and genomic knock-ins of constitutive expressionconstructs containing glmS*54, differing from the wild-type glmS protein(UniProt ID P17169) by an A39T, an R250C and a G472S mutation asdescribed by Deng et a1. (Biochimie 2006: 88, 419-429), GNA1 (UniProt IDP43577), the N-acetylglucosamine 2-epimerase (AGE) from Bacteroidesovatus (UniProt ID A7LVG6) and the N-acetylneuraminate synthase from N.meningitidis (UniProt ID E0NCD4), is further modified with a knock-in ofan N-acetylglucosamine β1,4-galactosyltransferase chosen from the listcomprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29,30, 31, 32, 33, 34, 37, 38 and 39.

Also, an E. coli K-12 MG1655 strain optimized for enhanced UDP-galactoseproduction with genomic knock-outs of the E. coli ushA and galT genesand with a genomic knock-in of a constitutive expression construct forthe UDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147) asdescribed in Example 1, was additionally mutated by a knock-out of theE. coli nagB gene and with a genomic knock-in of a constitutiveexpression construct of an N-acetylglucosamineβ1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37,38 and 39. The novel strains are evaluated for production of GlcNAc andLacNAc in a growth experiment according to the culture conditionsprovided in Example 1, in which the culture medium contains sucrose.Each strain is grown for 72 hours in multiple wells of a 96-well plate;afterwards the culture broth is harvested and the GlcNAc and LacNAc areanalyzed on UPLC.

Example 11: Production of Galactosylated Oligosaccharides in a ModifiedE. coli Host

An E. coli K-12 MG1655 strain optimized for enhanced UDP-galactoseproduction and capable of producing GlcNAc and LacNAc as described inExample 10, can additionally be transformed with an expression vectorcontaining a constitutive expression construct for anN-acetylglucosamine β1,3-galactosyltransferase chosen from the listcomprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9. The novel strain isevaluation for production of Gal-b14-(Galb13)-GlcNAc, containing twogalactose moieties linked beta-1,3 and beta-1,4 to GlcNAc, in additionto GlcNAc, LacNAc and LNB when evaluated in a growth experimentaccording to the culture conditions provided in Example 1, in which theculture medium contains sucrose.

Example 12: Production of Modified LacNAc in a E. coli Host

An E. coli K-12 MG1655 strain optimized for GDP-fucose production andcapable of produce GlcNAc and LacNAc as described in Example 9, canadditionally be transformed with an expression plasmid containing aconstitutive expression construct for theb1,3-N-acetyl-hexosaminyl-transferase LgtA from N. meningitidis (UniProtID Q9JXQ6). By subsequent action of the mutant glmS*54 (differing fromthe wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C anda G472S mutation), the homologous EcGlmM and EcGlmU and the heterologousLgtA (UniProt ID Q9JXQ6), the thus created strain is capable ofintracellularly converting fructose-6-phosphate into UDP-GlcNAc, and ofusing this UDP-GlcNAc to intracellularly modify LacNAc leading toproduction of GlcNAc-b1,3-Gal-b1,4-GlcNAc in whole broth samples whenevaluated in a growth experiment according to the culture conditionsprovided in Example 1, in which the culture medium contains sucrose. Thenovel strain is also capable of producing poly-LacNAc structures, i.e.,(Gal-b1,4-GlcNAc)n which are built of repeated N-acetyllactosamine thatare beta1,3-linked to each other by alternate activity of theN-acetylglucosamine β1,4-galactosyltransferase and LgtA expressed in thestrain.

An E. coli K-12 MG1655 strain optimized for UDP-galactose production andcapable of producing GlcNAc and LacNAc as described in Example 10, ismodified to constitutively express the UDP-GlcNAc epimerase wbpP fromPseudomonas aeruginosa (UniProt ID Q8KN66) and the glycosyltransferaselgtD from Haemophilus influenzae (UniProt ID A0A2X4DBP3). By subsequentaction of the mutant E. coli glmS*54, the homologous E. coli glmM andglmU and the P. aeruginosa wbpP, the cell is capable of intracellularlyconverting fructose-6-phosphate into UDP-GalNAc vithe intermediatecompounds glucosamine-6-phosphate, glucosamine-1-phosphate andUDP-GlcNAc. By subsequent action of the newly expressed LgtD enzyme, thenovel strain is capable of modifying the intracellularly produced LacNAcwith GalNAc, producing GalNAc-b1,3-Gal-b1,4-GlcNAc in whole brothsamples when evaluated in a growth experiment according to the cultureconditions provided in Example 1, in which the culture medium contains30 g/L sucrose.

Example 13: Production of LNB in a Modified E. coli Host

In a next experiment, an E. coli K-12 MG1655 strain optimized forGDP-fucose production was modified with genomic knock-ins ofconstitutive expression constructs for glmS*54 (differing from thewild-type glmS protein (UniProt ID P17169) by an A39T, an R250C and aG472S mutation) and GNA1 from S. cerevisiae (UniProt ID P43577) tointracellularly produce GlcNAc. In a next step, the mutant strain wasfurther modified for constitutive expression of the N-acetylglucosamineβ1,3-galactosyltransferase from C. glutamicum with SEQ ID NO: 3, eitherfrom plasmid or from a genomic knock-in. The novel strains wereevaluated in a growth experiment according to the culture conditionsprovided in Example 1. Table 2 shows the production of LNB (g/L) inwhole broth samples from each of the mutant strains, taken after 72hours of cultivation in minimal medium with 30 g/L sucrose. The datademonstrates that both novel strains produced LNB in whole brothsamples, independent of how the N-acetylglucosamineβ1,3-galactosyltransferase gene was presented to the strain.

TABLE 2 Production of LNB (g/L) in whole broth samples taken from mutantE. coli strains after 72 hours of cultivation in minimal mediumcomprising 30 g/L sucrose Expression of a transcriptional unit for theN- acetylglucosamine β1,3-galactosyltransferase with LNB (g/L) SEQ IDNO: 3 (± sd) From genomic knock-in 0.63 (±0.12) From an expressionplasmid 2.81 (±0.11)

Example 14: Production of Galactosylated LNB in a Modified E. coli Host

An E. coli K-12 MG1655 strain optimized for enhanced production ofUDP-galactose as described in Example 1 was additionally mutated by aknock-out of the E. coli nagB gene and was further modified forconstitutive expression of the N-acetylglucosamineβ1,3-galactosyltransferase from C. glutamicum with SEQ ID NO: 3, eitherfrom plasmid or from a genomic knock-in. Both novel strains produce LNBin whole broth samples when evaluated in a growth experiment accordingto the culture conditions provided in Example 1, in which the culturemedium contains sucrose. When the novel LNB production strains areadditionally transformed with expression constructs forgalactosyltransferases each of the novel strains produces GlcNAc and LNBtogether with galactosylated LNB forms in whole broth samples, whenevaluated in a growth experiment according to the culture conditionsprovided in Example 1, in which the culture medium contains sucrose.

Example 15: Fermentative Production of LacNAc with a Mutant E. coli Host

A mutant E. coli K-12 MG1655 strain optimized for GDP-fucose productionas described in Example 1 and modified to produce GlcNAc and LacNAc bygenomic knock-ins of constitutive transcriptional units for glmS*54(differing from the wild-type glmS protein (UniProt ID P17169) by anA39T, an R250C and a G472S mutation) and GNA1 (UniProt ID P43577), wasevaluated in a fed-batch fermentation at 5 L bioreactor scale accordingto the conditions provided in Example 1. In this example, sucrose wasused as a carbon source. Regular samples were taken and the productionof LacNAc was measured, as described in Example 1.

Example 16: Production of LacNAc or LNB in a Modified E. coli Host whenGrown on Other Carbon Sources than Sucrose

Mutant E. coli strains modified for the production of GlcNAc and LacNAcas described in Examples 9 and 10 or mutant E. coli strains modified forthe production of GlcNAc and LNB as described in Example 13 and 14 arecapable of producing GlcNAc and LacNAc or GlcNAc and LNB, respectively,when evaluated in a growth experiment according to the cultureconditions provided in Example 1, in which the culture medium containsglycerol. The mutant strains are also capable of producing GlcNAc andLacNAc or GlcNAc and LNB, respectively, when evaluated in fed-batchfermentations at bioreactor scale, as described in Example 1, using anyone or more of but not limited to following carbon sources: glycerol,glucose, fructose, lactose, arabinose, maltotriose, sorbitol, xylose,rhamnose and mannose.

Example 17: Materials and Methods in Saccharomyces cerevisiae

Media: Strains are grown on Synthetic Defined yeast medium with CompleteSupplement Mixture (SD CSM) or CSM drop-out (SD CSM-Ura) containing 6.7g/L Yeast Nitrogen Base without amino acids (YNB w/o AA, Difco), 20 g/Lagar (Difco) (solid cultures), 22 g/L glucose monohydrate or 20 g/Llactose and 0.79 g/L CSM or 0.77 g/L CSM-Ura (MP Biomedicals).

Strains: Saccharomyces cerevisiae BY4742 created by Brachmann et a1.(Yeast (1998) 14:115-32) was used, available in the Euroscarf culturecollection. All mutant strains were created by homologous recombinationor plasmid transformation using the method of Gietz (Yeast 11:355-360,1995).

Plasmids: Yeast expression plasmid p2a_2μ (Chan 2013, Plasmid 70, 2-17)was used for expression of foreign genes in Saccharomyces cerevisiae.This plasmid contained an ampicillin resistance gene and a bacterialorigin of replication to allow for selection and maintenance in E. coli.The plasmid further contained the 2p yeast on and the Ura3 selectionmarker for selection and maintenance in yeast. In one example, the yeastexpression plasmid p2a_2μ can be modified to obtain the mutantfructose-6-phosphate aminotransferase glmS*54 from E. coli (differingfrom the wild-type glmS protein (UniProt ID P17169) by an A39T, an R250Cand a G472S mutation) as described in WO18122225, the glucosamine6-phosphate N-acetyltransferase GNA1 from S. cerevisiae (UniProt IDP43577), a phosphatase like any one or more of, e.g., the E. coli genescomprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA,YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb,YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonas putida,ScDOG1 from S. cerevisiae or BsAraL from Bacillus subtilis as describedin WO18122225. The modified plasmids can further be modified to obtainan N-acetylglucosamine b-1,3-galactosyltransferase chosen from the listcomprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and/or an N-acetylglucosamineb-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37,38 and 39.

In one example to produce GDP-fucose, a yeast expression plasmid likep2a_2μ_Fuc (Chan 2013, Plasmid 70, 2-17) is further modified withconstitutive transcriptional units for a lactose permease like, e.g.,LAC12 from K. lactis (UniProt ID P07921), a GDP-mannose 4,6-dehydrataselike, e.g., gmd from E. coli (UniProt ID P0AC88) and a GDP-L-fucosesynthase like, e.g., fcl from E. coli (UniProt ID P32055). The yeastexpression plasmid p2a_2μ_Fuc2 can be used as an alternative expressionplasmid of the p2a_2μ_Fuc plasmid comprising next to the ampicillinresistance gene, the bacterial ori, the 2μ yeast on and the Ura3selection marker constitutive transcriptional units for a lactosepermease like, e.g., LAC12 from K. lactis (UniProt ID P07921), a fucosepermease like, e.g., fucP from E. coli (UniProt ID P11551) and abifunctional enzyme with fucose kinase/fucose-1-phosphateguanylyltransferase activity like, e.g., fkp from Bacteroides fragilis(UniProt ID SUV40286.1). To further produce fucosylatedoligosaccharides, the p2a_2μ_Fuc and its variant the p2a_2μ_Fuc2,additionally contained a constitutive transcriptional unit for analpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori (GenBankNo. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like, e.g.,HpFucT from H. pylori (UniProt ID 030511).

In one example to produce UDP-galactose, a yeast expression plasmid canbe derived from the pRS420-plasmid series (Christianson et al., 1992,Gene 110: 119-122) containing the HIS3 selection marker and aconstitutive transcriptional unit for a UDP-glucose-4-epimerase like,e.g., galE from E. coli (UniProt ID P09147). This plasmid can be furthermodified with constitutive transcriptional units for a lactose permeaselike, e.g., LAC12 from K. lactis (UniProt ID P07921) and a galactosidebeta-1,3-N-acetylglucosaminyltransferase activity like, e.g., lgtA fromN. meningitidis (UniProt ID Q9JXQ6) to produce LN3. To further produceLN3-derived oligosaccharides like LNT, the mutant LN3 producing strainswere further modified with a constitutive transcriptional unit for anN-acetylglucosamine beta-1,3-galactosyltransferase chosen from the listcomprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9. To further produceLN3-derived oligosaccharides like LNnT, the mutant LN3 producing strainswere further modified with a constitutive transcriptional unit for anN-acetylglucosamine beta-1,4-galactosyltransferase chosen from the listcomprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29,30, 31, 32, 33, 34, 37, 38 and 39.

In one example to produce sialic acid and CMP-sialic acid, a yeastexpression plasmid can be derived from the pRS420-plasmid series(Christianson et al., 1992, Gene 110: 119-122) containing the TRP1selection marker and constitutive transcriptional units for one or morecopies of an L-glutamine-D-fructose-6-phosphate aminotransferase like,e.g., the mutant glmS*54 from E. coli (differing from the wild-type E.coli glmS, having UniProt ID P17169, by an A39T, an R250C and a G472Smutation as described by Deng et a1. (Biochimie 88, 419-29 (2006)), onephosphatase like any one or more of, e.g., the E. coli genes comprisingaphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX,YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL,YjjG, YrfG and YbiU or PsMupP from Pseudomonas putida, ScDOG1 from S.cerevisiae or BsAraL from Bacillus subtilis as described in WO18122225,an N-acetylglucosamine 2-epimerase like, e.g., AGE from B. ovatus(UniProt ID A7LVG6), an N-acetylneuraminate synthase like, e.g., fromNeisseria meningitidis (UniProt ID E0NCD4), and an N-acylneuraminatecytidylyltransferase like, e.g., the NeuA enzyme from C. jejuni (UniProtID Q93MP7), the NeuA enzyme from Haemophilus influenzae (GenBank No.AGV11798.1) or the NeuA enzyme from Pasteurella multocida (GenBank No.AMK07891.1). Optionally, a constitutive transcriptional unit comprisingone or more copies for a glucosamine 6-phosphate N-acetyltransferaselike, e.g., GNA1 from S. cerevisiae (UniProt ID P43577) was/were addedas well. To produce sialylated oligosaccharides, the plasmid furthercomprised constitutive transcriptional units for a lactose permeaselike, e.g., LAC12 from Kluyveromyces lactis (UniProt ID P07921), and oneor more copies of a beta-galactoside alpha-2,3-sialyltransferase like,e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-likepolypeptide consisting of amino acid residues 1 to 268 of UniProt IDQ9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity,NmeniST3 from N. meningitidis (GenBank No. ARC07984.1) or PmultST2 fromP. multocida subsp. multocida str. Pm70 (GenBank No. AAK02592.1), abeta-galactoside alpha-2,6-sialyltransferase like, e.g., PdST6 fromPhotobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptideconsisting of amino acid residues 108 to 497 of UniProt ID 066375 havingbeta-galactoside alpha-2,6-sialyltransferase activity orP-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1)or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues18 to 514 of UniProt ID A8QYL1 having beta-galactosidealpha-2,6-sialyltransferase activity, and/or analpha-2,8-sialyltransferase like, e.g., from M. musculus (UniProt IDQ64689).

Preferably, but not necessarily, the glycosyltransferase proteins and/orthe proteins involved in nucleotide-activated sugar synthesis wereN-terminally fused to a SUMOstar tag (e.g., obtained from pYSUMOstar,Life Sensors, Malvern, PA) to enhance their solubility.

Optionally, the mutant yeast strains were modified with a knock-ins of aconstitutive transcriptional unit encoding a chaperone protein like,e.g., Hsp31, Hsp32, Hsp33, Sno4, Kar2, Ssb1, Sse1, Sse2, Ssa1, Ssa2,Ssa3, Ssa4, Ssb2, Ecm10, Ssc1, Ssq1, Ssz1, Lhs1, Hsp82, Hsc82, Hsp78,Hsp104, Tcp1, Cct4, Cct8, Cct2, Cct3, Cct5, Cct6, or Cct7 (Gong et al.,2009, Mol. Syst. Biol. 5: 275).

Plasmids were maintained in the host E. coli DH5alpha (F⁻,phi80dlacZdeltaM15, delta(lacZYA-argF)U169, deoR, recAl, endAl,hsdR17(rk⁻, mk⁺), phoA, supE44, lambda⁻, thi-1, gyrA96, relA1) boughtfrom Invitrogen.

Heterologous and homologous expression: Genes that needed to beexpressed, be it from a plasmid or from the genome were syntheticallysynthetized with one of the following companies: DNA2.0, Gen9, IDT orTwist Bioscience.

Expression could be further facilitated by optimizing the codon usage tothe codon usage of the expression host. Genes were optimized using thetools of the supplier.

Cultivations conditions: In general, yeast strains were initially grownon SD CSM plates to obtain single colonies. These plates were grown for2-3 days at 30° C.

Starting from a single colony, a pre-culture was grown over night in 5mL at 30° C., shaking at 200 rpm. Subsequent 125 mL shake flaskexperiments were inoculated with 2% of this pre-culture, in 25 mL media.These shake flasks were incubated at 30° C. with an orbital shaking of200 rpm.

Gene expression promoters: Genes were expressed using syntheticconstitutive promoters, as described by Blazeck (Biotechnology andBioengineering, Vol. 109, No. 11, 2012).

Example 18: Production of GlcNAc and LacNAc; or GlcNAc and LNB in S.cerevisiae

Another example provides use of a eukaryotic organism, in the form of S.cerevisiae, for performing the disclosure. Using the strains, plasmidsand methods as described in Example 17, a mutant S. cerevisiae strain iscreated that produces GlcNAc and LacNAc. These modifications comprisethe addition of constitutive expression units for the mutantfructose-6-phosphate aminotransferase glmS*54 of E. coli (differing fromthe wild-type E. coli glmS, having UniProt ID P17169, by an A39T, anR250C and a G472S mutation), the glucosamine 6-phosphateN-acetyltransferase GNA1 of S. cerevisiae (UniProt ID P43577), onephosphatase chosen from the list comprising any one or more of the E.coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP,YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV,YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonasputida, ScDOG1 from S. cerevisiae and BsAraL from Bacillus subtilis asdescribed in WO18122225 and an N-acetylglucosamineb-1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37,38 and 39. The mutant S. cerevisiae strain is capable of growing onglucose or glycerol as carbon source, converting the carbon source intofructose-6-phosphate, which is then converted to GlcNAc and subsequentlyLacNAc, by activity of the novel expressed fructose-6-phosphateaminotransferase, glucosamine 6-phosphate N-acetyltransferase, thephosphatase and N-acetylglucosamine b-1,4-galactosyltransferase.

Pre-culture of the strain is made in 5 mL of the synthetic definedmedium SD-CSM containing 22 g/L glucose and grown at 30° C. as describedin Example 17. This pre-culture is then inoculated in 25 mL medium in ashake flask with 10 g/L glucose as sole carbon source and grown at 30°C. Regular samples are taken and the production of GlcNAc and LacNAc ismeasured as described in Example 1.

A similar yeast strain containing an N-acetylglucosamineb-1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs:3, 4, 6, 7, 8 and 9 instead of the N-acetylglucosamineb-1,4-galactosyltransferase is capable of producing GlcNAc and LNB in asimilar cultivation experiment.

Example 19: Enzymatic Production of Galactosylated Di- orOligosaccharides

Another example provides the use of N-acetylglucosamineb-1,3-galactosyltransferases and N-acetylglucosamineb-1,4-galactosyltransferases of the disclosure to synthesizegalactosylated di- or oligosaccharides. These enzymes are produced in acell-free expression system such as but not limited to the PURExpresssystem (NEB), or in a host organism such as but not limited toEscherichia coli or Saccharomyces cerevisiae, after which theabove-listed enzymes can be isolated and optionally further purified.

An N-acetylglucosamine b-1,3-galactosyltransferase or anN-acetylglucosamine b-1,4-galactosyltransferase, selected from the aboveenzyme extracts or purified enzymes are added to a reaction mixturetogether UDP-galactose and a buffering component such as Tris-HCl orHEPES and GlcNAc, GalNAc or a di- or oligosaccharide containing anon-reducing (terminal) GlcNAc or GalNAc as acceptor. The reactionmixture is then incubated at a certain temperature (for example, 37° C.)for a certain amount of time (for example, 24 hours), during which theacceptor will be galactosylated on GlcNAc or GalNAc. The resultinggalactosylated di- or oligosaccharide is then separated from thereaction mixture by methods known in the art. Further purification ofthe galactosylated di- or oligosaccharide can be performed if preferred.At the end of the reaction or after separation and/or purification, theproduction of the galactosylated di- or oligosaccharide is measured asdescribed in Example 1.

Example 20: RegEx Search for N-Acetylglucosamineb-1,3-Galactosyltransferase Genes Having PFAM Domain PF00535

A RegEx analysis was performed for the N-acetylglucosamineb-1,3-galactosyltransferase genes having PFAM domain PF00535 to findmembers comprising the sequence [AGPS]XXLN(X_(n))RXDXD with SEQ ID NO:1, wherein X is any amino acid except for the combination XX onpositions 2 and 3 that cannot be an FA, FS, YC or YS combination andwherein n is 12 to 17, or members comprising the sequencePXXLN(X_(n))RXDXD(X_(m))[FWY]XX[HKR]XX[NQST] with SEQ ID NO: 2, whereinX is any amino acid except for the combination XX on positions 2 and 3that cannot be an FA, FS, YC or YS combination and wherein n is 12 to 17and m is 100 to 115. To this end, all N-acetylglucosamineb-1,3-galactosyltransferase genes having PFAM domain PF00535 asannotated in the Pfam database version Pfam 33.1 (as released on Jun.11, 2020) were downloaded from the UniProt database (as released on Jul.3, 2020) and analyzed for the presence of the motifs according themethod as available on:towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2(as released on Apr. 6, 2019). Corresponding members from the RegExsearch comprised A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6,A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0,A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6,A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2,A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63,A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3.

Example 21: RegEx Searches for Other N-Acetylglucosamineb-1,3-Galactosyltransferase or N-Acetylglucosamineb-1,4-Galactosyltransferase Genes

A similar RegEx analysis as exemplified in Example 20 can be performedfor the N-acetylglucosamine b-1,3-galactosyltransferase genes havingPFAM domain IPR002659 to find members comprising the sequenceKT(Xn)[FY]XXKXDXD(Xm)[FHY]XXG(X, no A, G, S)(Xp)(X, no F, H, W,Y)[DE]D[ILV]XX[AG] with SEQ ID NO: 05, wherein X is any amino acid andwherein n is 13 to 16, m is 35 to 70 and p is 20 to 45. To this end, allN-acetylglucosamine b-1,3-galactosyltransferase genes having PFAM domainIPR002659 as annotated in the Pfam database version Pfam 33.1 (asreleased on Jun. 11, 2020) were downloaded from the UniProt database (asreleased on Jul. 3, 2020) and analyzed for the presence of the motifsaccording the method as available on:towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2(as released on 6 Apr. 2019).

Similarly, a RegEx analysis can be performed for the N-acetylglucosamineb-1,4-galactosyltransferase genes having PFAM domain PF01755 to findmembers comprising the sequenceEXXCXXSHX[AFILTY]LW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 10,wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75,or comprising the sequence EXXCXXSH[LR]VLW(Xn)EDD(Xm)[ACGST]XXY[ILMV]with SEQ ID NO: 11, wherein X is any amino acid and wherein n is 13 to15 and m is 50 to 75, or comprising the sequenceEXXCXXSH[VHI]SLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 12, whereinX is any amino acid and wherein n is 13 to 15 and m is 50 to 75, orcomprising the sequence EXXCXXSHYMLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQID NO: 13, wherein X is any amino acid and wherein n is 13 to 15 and mis 50 to 75, or comprising the sequence EXXCXXSHXX(X, noV)Y(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 14, wherein X is anyamino acid and wherein n is 13 to 15 and m is 50 to 75. To this end, allN-acetylglucosamine b-1,4-galactosyltransferase genes having PFAM domainPF01755 as annotated in the Pfam database version Pfam 33.1 (as releasedon Jun. 11, 2020) were downloaded from the UniProt database (as releasedon Jul. 3, 2020) and analyzed for the presence of the motifs accordingthe method as available on:towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2(as released on Apr. 6, 2019).

A RegEx analysis can also be performed for the N-acetylglucosamineb-1,4-galactosyltransferase genes having PFAM domain PF00535 to findmembers comprising the sequence R[KN]XXXXXXXGXXXX[FL](X, noV)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE] with SEQ ID NO: 24 wherein X is anyamino acid and wherein n is 50 to 75 and m is 10 to 30, or comprisingthe sequence R[KN]XXXXXXXGXXXX[FL](X, noV)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE](Xp)[FWY]XX[HKR]XX[NQST] with SEQ ID NO:25 wherein X is any amino acid and wherein n is 50 to 75, m is 10 to 30and p is 20 to 25. To this end, all N-acetylglucosamineb-1,4-galactosyltransferase genes having PFAM domain PF00535 asannotated in the Pfam database version Pfam 33.1 (as released on Jun.11, 2020) were downloaded from the UniProt database (as released on Jul.3, 2020) and analyzed for the presence of the motifs according themethod as available on:towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2(as released on Apr. 6, 2019).

A RegEx analysis can also be performed for the N-acetylglucosamineb-1,4-galactosyltransferase genes having PFAM domain PF02709 and nothaving PFAM domain PF00535 to find members comprising the sequence[FWY]XX[FWY](Xn)[FWY][GQ]X[DE]D with SEQ ID NO: 28 wherein X is anyamino acid except for the combination XX on positions 2 and 3 thatcannot be an IP or NL combination and wherein n is 21 to 26. To thisend, all N-acetylglucosamine b-1,4-galactosyltransferase genes havingPFAM domain PF02709 and not having PFAM domain PF00535 as annotated inthe Pfam database version Pfam 33.1 (as released on Jun. 11, 2020) weredownloaded from the UniProt database (as released on Jul. 3, 2020) andanalyzed for the presence of the motifs according the method asavailable on:towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2(as released on Apr. 6, 2019).

Finally, a RegEx analysis can be performed for the N-acetylglucosamineb-1,4-galactosyltransferase genes having PFAM PF03808 to find memberscomprising the sequence[ST][FHY]XN(Xn)DGXXXXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA with SEQ ID NO:35, wherein X is any amino acid and wherein n is 20 to 25, or comprisingthe sequence[ST][FHY]XN(Xn)DGXXXXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA(Xm)[HR]XG[FWY](Xp)GXGXXXQ[DE] with SEQ ID NO: 36, wherein X is any amino acid andwherein n is 20 to 25, m is 40 to 50 and p is 22 to 30. To this end, allN-acetylglucosamine b-1,4-galactosyltransferase genes having PFAM domainPF03808 as annotated in the Pfam database version Pfam 33.1 (as releasedon Jun. 11, 2020) were downloaded from the UniProt database (as releasedon Jul. 3, 2020) and analyzed for the presence of the motifs accordingthe method as available on:towardsdatascience.com/using-regular-expression-in-genetics-with-python-175e2b9395c2(as released on Apr. 6, 2019).

Example 22: Production of an Oligosaccharide Mixture Comprising 6′-SL,LacNAc, Sialylated LacNAc, LN3, Sialylated LN3, LNnT and LSTc with aModified E. coli Host

An E. coli K-12 strain MG1655 is modified for sialic acid production asdescribed in Example 1 comprising knock-outs of the E. coli nagA, nagB,nanA, nanT, nanE, nanK, LacZ, LacY and LacA genes and genomic knock-insof constitutive transcriptional units comprising genes encoding thelactose permease (LacY) from E. coli (UniProt ID P02920), the sialicacid transporter (nanT) from E. coli (UniProt ID P41036), the mutantL-glutamine-D-fructose-6-phosphate aminotransferase glmS*54 from E. coli(differing from the wild-type E. coli glmS, having UniProt ID P17169, byan A39T, an R250C and a G472S mutation), the glucosamine 6-phosphateN-acetyltransferase (GNA1) from S. cerevisiae (UniProt ID P43577), theN-acetylglucosamine 2-epimerase (AGE) from B. ovatus (UniProt IDA7LVG6), the N-acetylneuraminate synthase (NeuB) from C. jejuni (UniProtID Q93MP9), the sucrose transporter (CscB) from E. coli W (UniProt IDE0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417)and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt IDA0ZZH6). The thus obtained mutant E. coli strain producing sialic acidis further modified with a genomic knock-in of constitutivetranscriptional units to express the N-acylneuraminatecytidylyltransferase enzyme NeuA from C. jejuni (UniProt ID Q93MP7) andthe alpha-2,6-sialyltransferase PdbST from P. damselae (UniProt ID066375) to produce 6′-sialyllactose. In a next step, the mutant strainis further modified with genomic knock-ins comprising constitutivetranscriptional units for the galactosidebeta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis(UniProt ID Q9JXQ6) and an N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34,37, 38 and 39 to produce a mixture of oligosaccharides comprising 6′-SL,LacNAc, sialylated LacNAc, LN3, sialylated LN3, LNnT and LSTc(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc). The novel strains areevaluated in a growth experiment according to the culture conditionsprovided in Example 1, in which the culture medium contains sucrose andlactose. The strains are grown in four biological replicates in a96-well plate. After 72 hours of incubation, the culture broth isharvested, and the sugars are analyzed on UPLC.

Example 23: Production of an Oligosaccharide Mixture LN3, SialylatedLN3, LNT, LNB, Sialylated LNB, 3′-SL and LSTa with a Modified E. coliHost

An E. coli strain modified to produce sialic acid (Neu5Ac) as describedin Example 22 is further modified with a genomic knock-in ofconstitutive transcriptional units to express the N-acylneuraminatecytidylyltransferase enzyme NeuA from C. jejuni (UniProt ID Q93MP7) andthe alpha-2,3-sialyltransferase PmultST3 from P. multocida (UniProt IDQ9CLP3) to produce 3′-siayllactose. In a next step, the mutant strain isfurther modified with genomic knock-ins comprising constitutivetranscriptional units for the galactosidebeta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis(UniProt ID Q9JXQ6) and an N-acetylglucosaminebeta-1,3-galactosyltransferase chosen from the list comprising SEQ IDNOs: 3, 4, 6, 7, 8 and 9 to produce a mixture of oligosaccharidescomprising LN3,3′-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc),LNT, LNB, sialylated LNB, 3′-SL and LSTa(Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc). The novel strains areevaluated in a growth experiment per the culture conditions of Example1, in which the culture medium contains sucrose and lactose. The strainsare grown in four biological replicates in a 96-well plate. After 72hours of incubation, the culture broth is harvested, and the sugars areanalyzed on UPLC.

Example 24: Production of Fucosylated LNB Forms in Modified E. coliHosts

An E. coli K-12 MG1655 mutant strain optimized for GDP-fucose productionand growth on sucrose as described in Example 1, is further modifiedwith a knock-out of the E. coli nagA and nagB genes and with a genomicknock-in of a constitutive expression construct of anN-acetylglucosamine β1,3-galactosyltransferase chosen from the listcomprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9. In a next step, cells of themutant strain are transformed with an expression vector comprisingconstitutive transcriptional units of the mutant glmS*54 from E. coli(differing from the wild-type glmS protein (UniProt ID P17169) by anA39T, an R250C and a G472S mutation) and GNA1 from S. cerevisiae(UniProt ID P43577). In a further step, the novel strain is transformedwith a second compatible expression vector comprising a constitutivetranscriptional unit for the a1,2-fucosyltransferase HpFutC (GenBank NO.AAD29863.1) and/or the a1,3-fucosyltransferase HpFucT (UniProt ID030511). The novel strains are evaluated for production of GlcNAc, LNB,and fucosylated LNB forms (2′FLNB, 4′FLNB and/or difucosylated LNB) in agrowth experiment according to the culture conditions provided inExample 1, in which the culture medium contains sucrose.

Example 25: Production of Fucosylated LNB and Lactose Forms in ModifiedE. coli Hosts

The mutant E. coli strains described in Example 24 can further bemodified comprising genomic knock-out of the E. coli genes galT, ushA,ldhA, LacZ, LacY and LacA and a genomic knock-in of a constitutivetranscriptional unit for the lactose permease LacY from E. coli (UniProtID P02920).

When the novel mutant strains are cultivated in a growth experimentaccording to the culture conditions provided in Example 1, in which theculture medium contains sucrose and lactose, the strains can beevaluated for the production of GlcNAc, LNB and fucosylated LNB andlactose forms like 2′-FLNB, 4-FLNB, 2′FL, 3-FL and/or DiFL.

Example 26: Production of a Neutral Oligosaccharide Mixture ComprisingFucosylated Structures in Modified E. coli Hosts

An E. coli K-12 MG1655 mutant strain optimized for GDP-fucose productionand growth on sucrose as described in Example 1, is further modifiedwith a knock-out of the E. coli nagA and nagB genes and with a genomicknock-in of a constitutive expression construct of the galactosidebeta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis(GenBank: AAM33849.1) and an N-acetylglucosamineβ1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs:3, 4, 6, 7, 8 and 9. In a next step, cells of the mutant strain aretransformed with an expression vector comprising constitutivetranscriptional units of the mutant glmS*54 from E. coli (differing fromthe wild-type glmS protein (UniProt ID P17169) by an A39T, an R250C anda G472S mutation) and GNA1 from S. cerevisiae (UniProt ID P43577). In afurther step, the novel strain is transformed with a second compatibleexpression vector comprising a constitutive transcriptional unit for thea1,2-fucosyltransferase HpFutC (GenBank NO. AAD29863.1).

The novel mutant strain is evaluated for the production of a neutraloligosaccharide mixture comprising LNB, fucosylated LNB, 2′FL, DiFL, LN3(lacto-N-triose), LNT (lacto-N-tetraose) and LNFP-I(Lacto-N-fucopentaose I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) ina growth experiment according to the culture conditions provided inExample 1, in which the culture medium contains sucrose and lactose.

Example 27: Production of a Neutral Oligosaccharide Mixture ComprisingFucosylated Structures in Modified E. coli Hosts

An E. coli K-12 MG1655 mutant strain optimized for GDP-fucose productionand growth on sucrose as described in Example 1, is further modifiedwith a knock-out of the E. coli nagA and nagB genes and with a genomicknock-in of a constitutive expression construct of the galactosidebeta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis(GenBank: AAM33849.1) and an N-acetylglucosamineβ1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37,38 and 39. In a next step, cells of the mutant strain are transformedwith an expression vector comprising constitutive transcriptional unitsof the mutant glmS*54 from E. coli (differing from the wild-type glmSprotein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation)and GNA1 from S. cerevisiae (UniProt ID P43577). In a further step, thenovel strain is transformed with a second compatible expression vectorcomprising a constitutive transcriptional unit for thea1,3-fucosyltransferase HpFucT (UniProt ID 030511).

The novel mutant strain is evaluated for the production of a neutraloligosaccharide mixture comprising LacNAc, fucosylated LacNAc, 3-FL, LN3(lacto-N-triose), LNnT (lacto-N-tetraose) and LNFP-III(Lacto-N-fucopentaose III, Gal-b1,4-(Fuc-a1,3)-GlcNAc-b1,3-Gal-b1,4-Glc)in a growth experiment according to the culture conditions provided inExample 1, in which the culture medium contains sucrose and lactose.

Example 28: Production of LacNAc and 3-FLN in Modified E. coli Hosts

An E. coli K-12 MG1655 mutant strain optimized for GDP-fucose productionas described in Example 1, is further modified with a knock-out of theE. coli nagA and nagB genes and with a genomic knock-in of aconstitutive expression construct of an N-acetylglucosamineβ1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37,38 and 39. In a next step, cells of the mutant strain are transformedwith an expression vector comprising constitutive transcriptional unitsof the mutant glmS*54 from E. coli (differing from the wild-type glmSprotein (UniProt ID P17169) by an A39T, an R250C and a G472S mutation)and GNA1 from S. cerevisiae (UniProt ID P43577). In a further step, thenovel strain is transformed with a second compatible expression vectorcomprising a constitutive transcriptional unit for thea1,3-fucosyltransferase HpFucT (UniProt ID 030511). The novel strainsare evaluated for production of GlcNAc, LacNAc and 3-FLN(Gal-b1,4-(Fuc-a1,3)-GlcNAc) in a growth experiment according to theculture conditions provided in Example 1, in which the culture mediumcontains sucrose.

Example 29: Production of the T-Disaccharide (Gal-b1,3-GalNAc) in aModified E. Coli Host

An E. coli K-12 MG1655 strain is optimized for enhanced UDP-galactoseproduction as described in Example 1 with genomic knock-outs of the E.coli ushA and galT genes and with a genomic knock-in of a constitutiveexpression construct for the UDP-glucose4-epimerase (galE) of E. coli(UniProt ID P09147). In a next step the strain is additionally mutatedby a knock-out of the E. coli nagB gene and with a genomic knock-in of aconstitutive expression construct of an N-acetylglucosamineβ1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs:3, 4, 6, 7, 8 and 9. The novel strain is evaluated for the production ofthe T-disaccharide (Gal-b1,3-GalNAc) when cultivated in a growthexperiment according to the culture conditions provided in Example 1, inwhich the culture medium contains glucose and GalNAc.

Example 30: Production of Gal-b1,4-GalNAc in a Modified E. coli Host

An E. coli K-12 MG1655 strain is optimized for enhanced UDP-galactoseproduction as described in Example 1 with genomic knock-outs of the E.coli ushA and galT genes and with a genomic knock-in of a constitutiveexpression construct for the UDP-glucose4-epimerase (galE) of E. coli(UniProt ID P09147). In a next step, the strain is additionally mutatedby a knock-out of the E. coli nagB gene and with a genomic knock-in of aconstitutive expression construct of an N-acetylglucosamineβ1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37,38 and 39. In a final step, the mutant strain is adapted for growth onsucrose via genomic knock-ins of constitutive transcriptional unitscontaining the sucrose transporter CscB from E. coli W (UniProt IDE0IXR1), the fructose kinase Frk originating from Z. mobilis (UniProt IDQ03417) and the sucrose phosphorylase BaSP from B. adolescentis (UniProtID A0ZZH6). The strain is evaluated for the production ofGal-b1,4-GalNAc when cultivated in a growth experiment according to theculture conditions provided in Example 1, in which the culture mediumcontains sucrose and GalNAc.

Example 31: Production of LN3 and LNT in a Modified E. coli Host

An E. coli K-12 strain MG1655 is modified as described in Example 1comprising knock-outs of the E. coli nagB, galT, ushA, agp, ldhA, LacZ,LacY and LacA genes and genomic knock-ins of constitutivetranscriptional units comprising the genes encoding the lactose permease(LacY) from E. coli (UniProt ID P02920), the sucrose transporter (CscB)from E. coli W (UniProt ID EOIXR1), the fructose kinase (Frk) from Z.mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B.adolescentis (UniProt ID A0ZZH6). In a next step, the mutant E. colistrain is modified for LN3 production with a genomic knock-in of aconstitutive transcriptional unit for the galactosidebeta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis(GenBank: AAM33849.1). In a further step, the mutant strain is modifiedfor LNT production with a genomic knock-in of a constitutivetranscriptional unit for an N-acetylglucosamineβ1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs:3, 4, 6, 7, 8, 9, and the polypeptides with UniProt ID A0A354SD93,A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4,T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0,A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0,A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8,A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7,A0A1G9SAW9, E3HB28 and A0A538TXM3. The novel strain is evaluated forproduction of LN3 and LNT in a growth experiment according to theculture conditions provided in Example 1, in which the culture mediumcontains 30 g/L sucrose and 20 g/L lactose. The strain is grown in fourbiological replicates in a 96-well plate. After 72 hours of incubation,the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 32: Production of LN3 and LNnT in a Modified E. coli Host

An E. coli K-12 strain MG1655 is modified as described in Example 1comprising knock-outs of the E. coli nagB, galT, ushA, agp, ldhA, LacZ,LacY and LacA genes and genomic knock-ins of constitutivetranscriptional units comprising the genes encoding the lactose permease(LacY) from E. coli (UniProt ID P02920), the sucrose transporter (CscB)from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z.mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B.adolescentis (UniProt ID A0ZZH6). In a next step, the mutant E. colistrain is modified for LN3 production with a genomic knock-in of aconstitutive transcriptional unit for the galactosidebeta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis(GenBank: AAM33849.1). In a further step, the mutant strain is modifiedfor LNT production with a genomic knock-in of a constitutivetranscriptional unit for an N-acetylglucosamineβ1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37,38, 39 and the N-acetylglucosamine b-1,4-galactosyltransferasepolypeptides identified as described by Example 21. The novel strain isevaluated for production of oligosaccharides comprising LN3, LNnT,lacto-N-neohexaose (LNnH) and para-lacto-N-neohexaose (pLNH) in a growthexperiment according to the culture conditions provided in Example 1, inwhich the culture medium contains 30 g/L sucrose and 20 g/L lactose. Thestrain is grown in four biological replicates in a 96-well plate. After72 hours of incubation, the culture broth is harvested, and the sugarsare analyzed on UPLC.

Example 33: Material and Methods Bacillus subtilis

Media: Two different media are used, namely a rich Luria Broth (LB) anda minimal medium for shake flask (MMsf). The minimal medium uses a traceelement mix.

Trace element mix consisted of 0.735 g/L CaCl2·2H2O, 0.1 g/L MnCl2.2H2O,0.033 g/L CuCl2·2H2O, 0.06 g/L CoCl2·6H2O, 0.17 g/L ZnCl2, 0.0311 g/LH3B04, 0.4 g/L Na2EDTA·2H₂O and 0.06 g/L Na2MoO4. The Fe-citratesolution contained 0.135 g/L FeCl3.6H2O, 1 g/L Na-citrate (Hoch 1973PMC1212887).

The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco,Erembodegem, BE), 0.5% yeast extract (Difco) and 0.5% sodium chloride(VWR. Leuven, BE). Luria Broth agar (LBA) plates consisted of the LBmedia, with 12 g/L agar (Difco, Erembodegem, BE) added.

The minimal medium for the shake flasks (MMsf) experiments contained2.00 g/L (NH4)2SO4, 7.5 g/L KH2PO4, 17.5 g/L K2HPO4, 1.25 g/LNa-citrate, 0.25 g/L MgSO4.7H2O, 0.05 g/L tryptophan, from 10 up to 30g/L glucose or another carbon source including but not limited tofructose, maltose, sucrose, glycerol and maltotriose when specified inthe examples, 10 ml/L trace element mix and 10 ml/L Fe-citrate solution.The medium was set to a pH of 7 with 1M KOH. Depending on the experimentsialic acid or lactose could be added as a precursor.

Complex medium, e.g., LB, was sterilized by autoclaving (121° C., 21min.) and minimal medium by filtration (0.22 μm Sartorius). Whennecessary, the medium was made selective by adding an antibiotic (e.g.,zeocin (20 mg/L)).

Strains, plasmids and mutations: B. subtilis 168, available at BacillusGenetic Stock Center (Ohio, USA).

Plasmids for gene deletion via Cre/lox are constructed as described byYan et a1. (Appl. & Environm. Microbial., September 2008, p 5556-5562).Gene disruption is done via homologous recombination with linear DNA andtransformation via electroporation as described by Xue et a1. (J.Microb. Meth. 34 (1999) 183-191). The method of gene knockouts isdescribed by Liu et a1. (Metab. Engine. 24 (2014) 61-69). This methoduses 1000 bp homologies up- and downstream of the target gene.

Integrative vectors as described by Popp et a1. (Sci. Rep., 2017, 7,15158) are used as expression vector and could be further used forgenomic integrations if necessary. A suitable promoter for expressioncan be derived from the part repository (iGem): sequence id:Bba_K143012, Bba_K823000, Bba_K823002 or Bba_K823003. Cloning can beperformed using Gibson Assembly, Golden Gate assembly, Cliva assembly,LCR or restriction ligation.

In an example for the production of LNB, Bacillus subtilis mutantstrains are modified with genomic knock-ins comprising constitutivetranscriptional units for the mutant glmS*54 from E. coli (differingfrom the wild-type E. coli glmS, having UniProt ID P17169, by an A39T,an R250C and a G472S mutation as described by Deng et a1. (Biochimie 88,419-29 (2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1from S. cerevisiae (UniProt ID P43577), one phosphatase chosen from thelist comprising any one or more of the E. coli genes comprising aphA,Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC,YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG,YrfG and YbiU or PsMupP from Pseudomonas putida, DOG1 from S. cerevisiaeor AraL from Bacillus subtilis as described in WO18122225 and anN-acetylglucosamine beta-1,3-galactosyltransferase chosen from the listcomprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides withUniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8,T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500,A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9,A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6,N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2,A0A5C5Y5M7, A0A1G9SAW9, E311B28 and A0A538TXM3.

In an example for the production of LacNAc, Bacillus subtilis mutantstrains are modified with genomic knock-ins comprising constitutivetranscriptional units for the mutant glmS*54 from E. coli (differingfrom the wild-type E. coli glmS, having UniProt ID P17169, by an A39T,an R250C and a G472S mutation as described by Deng et a1. (Biochimie 88,419-29 (2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1from S. cerevisiae (UniProt ID P43577), one phosphatase chosen from thelist comprising any one or more of the E. coli genes comprising aphA,Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC,YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG,YrfG and YbiU or PsMupP from Pseudomonas putida, DOG1 from S. cerevisiaeor AraL from Bacillus subtilis as described in WO18122225 and anN-acetylglucosamine beta-1,4-galactosyltransferase chosen from the listcomprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29,30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamineb-1,4-galactosyltransferase polypeptides identified as described inExample 21. To further fucosylate the LNB or LacNAc, the LNB or LacNAcproducing strains are further modified with a constitutivetranscriptional unit for an alpha-1,2-fucosyltransferase like, e.g.,HpFutC from H. pylori (GenBank No. AAD29863.1) and/or analpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProtID 030511).

In an example for the production of lactose-based oligosaccharides,Bacillus subtilis mutant strains are created to contain a gene codingfor a lactose importer (such as, e.g., the E. coli lacY with UniProt IDP02920). For 2′FL, 3FL and diFL production, an alpha-1,2—and/oralpha-1,3-fucosyltransferase expression construct is additionally addedto the strains.

In an example for the production of lacto-N-triose (LNT-II, LN3,GlcNAc-b1,3-Gal-b1,4-Glc), the B. subtilis strain is modified with agenomic knock-in of constitutive transcriptional units comprising alactose importer (such as, e.g., the E. coli lacY with UniProt IDP02920) and a galactoside beta-1,3-N-acetylglucosaminyltransferase like,e.g., LgtA from N. meningitidis (GenBank: AAM33849.1). For LNTproduction, the LN3 producing strain is further modified with aconstitutive transcriptional unit for an N-acetylglucosaminebeta-1,3-galactosyltransferase chosen from the list comprising SEQ IDNOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDsA0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3,U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8,A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3,A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3,A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2,A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3, that can be delivered tothe strain either via genomic knock-in or from an expression plasmid.For LNnT production, the LN3 producing strain is further modified with aconstitutive transcriptional unit for an N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34,37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferasepolypeptides identified as described in Example 21. To furtherfucosylate the LN3, LNT or LNnT, the mutant strains are further modifiedwith a constitutive transcriptional unit for analpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori (GenBankNo. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like, e.g.,HpFucT from H. pylori (UniProt ID 030511).

In an example for sialic acid production, a mutant B. subtilis strain iscreated by overexpressing the native fructose-6-P-aminotransferase(UniProt ID P0CI73) to enhance the intracellular glucosamine-6-phosphatepool. Further on, the enzymatic activities of the genes nagA, nagB andgamA are disrupted by genetic knockouts and aglucosamine-6-P-aminotransferase from S. cerevisiae (UniProt ID P43577),an N-acetylglucosamine-2-epimerase from B. ovatus (UniProt ID A7LVG6)and an N-acetylneuraminate synthase from C. jejuni (UniProt ID Q93MP9)are overexpressed on the genome. To allow sialylated oligosaccharideproduction, the sialic acid producing strain is further modified withexpression constructs comprising an N-acylneuraminatecytidylyltransferase like, e.g., the NeuA enzyme from C. jejuni (UniProtID Q93MP7), the NeuA enzyme from H. influenzae (GenBank No. AGV 11798.1)or the NeuA enzyme from P. multocida (GenBank No. AMK07891.1), and oneor more copies of a beta-galactoside alpha-2,3-sialyltransferase like,e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-likepolypeptide consisting of amino acid residues 1 to 268 of UniProt IDQ9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity,NmeniST3 from N. meningitidis (GenBank No. ARC07984.1) or PmultST2 fromP. multocida subsp. multocida str. Pm70 (GenBank No. AAK02592.1), abeta-galactoside alpha-2,6-sialyltransferase like, e.g., PdST6 fromPhotobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptideconsisting of amino acid residues 108 to 497 of UniProt ID 066375 havingbeta-galactoside alpha-2,6-sialyltransferase activity orP-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1)or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues18 to 514 of UniProt ID A8QYL1 having beta-galactosidealpha-2,6-sialyltransferase activity, and/or analpha-2,8-sialyltransferase like, e.g., from M. musculus (UniProt IDQ64689). In an example for the production of lactose-based sialylatedoligosaccharides, the Bacillus subtilis mutant strains are furthermodified with a constitutive transcriptional unit for a lactose importer(such as, e.g., the E. coli lacY with UniProt ID P02920).

For growth on sucrose, the mutant strains can additionally be modifiedwith genomic knock-ins of constitutive transcriptional units comprisingthe sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), thefructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and thesucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6).

Heterologous and homologous expression: Genes that needed to beexpressed, be it from a plasmid or from the genome were syntheticallysynthetized with one of the following companies: DNA2.0, Gen9, TwistBiosciences or IDT.

Expression could be further facilitated by optimizing the codon usage tothe codon usage of the expression host. Genes were optimized using thetools of the supplier.

Cultivation conditions: A pre-culture of 96-well microtiter plateexperiments was started from a cryovial or a single colony from an LBplate, in 150 μL LB and was incubated overnight at 37° C. on an orbitalshaker at 800 rpm. This culture was used as inoculum for a 96-wellsquare microtiter plate, with 400 μL MMsf medium by diluting 400×. Eachstrain was grown in multiple wells of the 96-well plate as biologicalreplicates. These final 96-well culture plates were then incubated at37° C. on an orbital shaker at 800 rpm for 72 hours, or shorter, orlonger. At the end of the cultivation experiment samples were taken fromeach well to measure the supernatant concentration (extracellular sugarconcentrations, after 5 min. spinning down the cells), or by boiling theculture broth for 15 min. at 90° C. or for 60 min. at 60° C. beforespinning down the cells (=whole broth concentration, intra- andextracellular sugar concentrations, as defined herein).

Also, a dilution of the cultures was made to measure the optical densityat 600 nm. The cell performance index or CPI was determined by dividingthe oligosaccharide concentrations by the biomass, in relativepercentages compared to a reference strain. The biomass is empiricallydetermined to be approximately 1/3rd of the optical density measured at600 nm.

Example 34: Production of 2′FLNB with a Modified B. subtilis Strain

A B. subtilis strain is first modified for LNB production and growth onsucrose by genomic knock-out of the nagB, glmS and gamA genes andgenomic knock-ins of constitutive transcriptional units comprising genesencoding the native fructose-6-P-aminotransferase (UniProt ID P0CI73),the mutant glmS*54 from E. coli (differing from the wild-type E. coliglmS, having UniProt ID P17169, by an A39T, an R250C and a G472Smutation as described by Deng et a1. (Biochimie 88, 419-29 (2006)), theglucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae(UniProt ID P43577), the phosphatase AraL from B. subtilis (UniProt IDP94526), an N-acetylglucosamine beta-1,3-galactosyltransferase chosenfrom the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and thepolypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10,A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3,A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259,A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1,A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2,F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 andA0A538TXM3, the sucrose transporter (CscB) from E. coli W (UniProt IDEOIXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417)and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt IDA0ZZH6). In a next step, the LNB producing strain is transformed with anexpression plasmid comprising a constitutive transcriptional unit forthe alpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori(GenBank No. AAD29863.1).

The novel strain is evaluated for the production 2′FLNB in a growthexperiment on MMsf medium lacking a precursor according to the cultureconditions provided in Example 33. After 72 hours of incubation, theculture broth is harvested, and the sugars are analyzed on UPLC.

Example 35: Production of an Oligosaccharide Mixture Comprising 6′-SL,LacNAc, Sialylated LacNAc, LN3, Sialylated LN3, LNnT and LSTc with aModified B. subtilis Strain

In a first step, a B. subtilis strain is modified for sialic acidproduction with genetic knockouts of the nagA, nagB and gamA genes andwith genomic knock-ins of constitutive transcriptional units comprisinggenes encoding the native fructose-6-P-aminotransferase (UniProt IDP0CI73), a glucosamine-6-P-aminotransferase from S. cerevisiae (UniProtID P43577), an N-acetylglucosamine-2-epimerase from B. ovatus (UniProtID A7LVG6) and an N-acetylneuraminate synthase from C. jejuni (UniProtID Q93MP9). In a next step, the mutant strain is further modified withgenomic knock-ins of constitutive transcriptional units comprising genesencoding the N-acylneuraminate cytidylyltransferase NeuA from C. jejuni(UniProt ID Q93MP7), and the alpha-2,6-sialyltransferase PdbST from P.damselae (UniProt ID 066375) to produce 6′-sialyllactose. In a nextstep, the mutant strain is further modified with genomic knock-inscomprising constitutive transcriptional units for the galactosidebeta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis(UniProt ID Q9JXQ6) and an N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34,37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferasepolypeptides identified as described in Example 21. The novel strain isevaluated for production of a mixture of oligosaccharides comprising6′-SL, LacNAc, sialylated LacNAc, LN3, sialylated LN3, LNnT and LSTc(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc) in a growth experimenton MMsf medium containing lactose as precursor according to the cultureconditions provided in Example 33. After 72 hours of incubation, theculture broth is harvested, and the sugars are analyzed on UPLC.

Example 36: Production of Gal-b1,4-GalNAc with a Modified B. subtilisStrain

A B. subtilis strain is modified with a genomic knock-in of aconstitutive expression construct for the UDP-glucose4-epimerase (galE)of E. coli (UniProt ID P09147), an N-acetylglucosamineβ1,4-galactosyltransferase chosen from the list comprising 15, 16, 17,18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39and the N-acetylglucosamine b-1,4-galactosyltransferase polypeptidesidentified as described in Example 21, the sucrose transporter CscB fromE. coli W (UniProt ID EOIXR1), the fructose kinase Frk originating fromZ. mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP fromB. adolescentis (UniProt ID A0ZZH6). The novel strain is evaluated forthe production of Gal-b1,4-GalNAc when cultivated in a growth experimentaccording to the culture conditions provided in Example 33, in which theculture medium contains sucrose and GalNAc.

Example 37: Production of Gal-b1,3-GalNAc with a Modified B. subtilisStrain

A B. subtilis strain is modified with a genomic knock-in of aconstitutive expression construct for the UDP-glucose4-epimerase (galE)of E. coli (UniProt ID P09147), an N-acetylglucosamineβ1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs:3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93,A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4,T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0,A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0,A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8,A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7,A0A1G9SAW9, E3HB28 and A0A538TXM3, the sucrose transporter CscB from E.coli W (UniProt ID EOIXR1), the fructose kinase Frk originating from Z.mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B.adolescentis (UniProt ID A0ZZH6). The novel strain is evaluated for theproduction of Gal-b1,3-GalNAc when cultivated in a growth experimentaccording to the culture conditions provided in Example 33, in which theculture medium contains sucrose and GalNAc.

Example 38: Material and Methods Corynebacterium glutamicum

Media: Two different media are used, namely a rich tryptone-yeastextract (TY) medium and a minimal medium for shake flask (MMsf). Theminimal medium uses a 1000× stock trace element mix.

Trace element mix consisted of 10 g/L CaCl2), 10 g/L FeSO4·7H2O, 10 g/LMnSO4·H2O, 1 g/L ZnSO4·7H2O, 0.2 g/L CuSO4, 0.02 g/L NiCl2·6H2O, 0.2 g/Lbiotin (pH 7.0) and 0.03 g/L protocatechuic acid.

The minimal medium for the shake flasks (MMsf) experiments contained 20g/L (NH4)2SO4, 5 g/L urea, 1 g/L KH2PO4, 1 g/L K2HPO4, 0.25 g/LMgSO4.7H2O, 42 g/L MOPS, from 10 up to 30 g/L glucose or another carbonsource including but not limited to fructose, maltose, sucrose, glyceroland maltotriose when specified in the examples and 1 ml/L trace elementmix. Depending on the experiment lactose and/or sialic acid could beadded as precursor(s).

The TY medium consisted of 1.6% tryptone (Difco, Erembodegem, BE), 1%yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, BE). TYagar (TYA) plates consisted of the TY media, with 12 g/L agar (Difco,Erembodegem, BE) added.

Complex medium, e.g., TY, was sterilized by autoclaving (121° C., 21min.) and minimal medium by filtration (0.22 μm Sartorius). Whennecessary, the medium was made selective by adding an antibiotic (e.g.,kanamycin, ampicillin).

Strains and mutations: Corynebacterium glutamicum ATCC 13032, availableat the American Type Culture Collection.

Integrative plasmid vectors based on the Cre/loxP technique as describedby Suzuki et a1. (Appl. Microbiol. Biotechnol., 2005 April,67(2):225-33) and temperature-sensitive shuttle vectors as described byOkibe et a1. (Journal of Microbiological Methods 85, 2011, 155-163) areconstructed for gene deletions, mutations and insertions. Suitablepromoters for (heterologous) gene expression can be derived from Yim eta1. (Biotechnol. Bioeng., 2013 November, 110(11):2959-69). Cloning canbe performed using Gibson Assembly, Golden Gate assembly, Clivaassembly, LCR or restriction ligation.

In an example for the production of LNB, the C. glutamicum strain ismodified with a genomic knock-in of constitutive expression unitscomprising the mutant glmS*54 from E. coli (differing from the wild-typeE. coli glmS, having UniProt ID P17169, by an A39T, an R250C and a G472Smutation as described by Deng et a1. (Biochimie 88, 419-29 (2006)), theglucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae(UniProt ID P43577), one phosphatase chosen from the list comprising anyone or more of the E. coli genes comprising aphA, Cof, HisB, OtsB, SurE,Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph,SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupPfrom P. putida, ScDOG1 from S. cerevisiae or BsAraL from B. subtilis asdescribed in WO18122225 and an N-acetylglucosaminebeta-1,3-galactosyltransferase chosen from the list comprising SEQ IDNOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDsA0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3,U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8,A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3,A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3,A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2,A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3.

In an example for the production of LacNAc, the C. glutamicum strain ismodified with a genomic knock-in of constitutive expression unitscomprising the mutant glmS*54 from E. coli (differing from the wild-typeE. coli glmS, having UniProt ID P17169, by an A39T, an R250C and a G472Smutation as described by Deng et a1. (Biochimie 88, 419-29 (2006)), theglucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae(UniProt ID P43577), one phosphatase chosen from the list comprising anyone or more of the E. coli genes comprising aphA, Cof, HisB, OtsB, SurE,Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph,SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupPfrom Pseudomonas putida, ScDOG1 from S. cerevisiae or BsAraL fromBacillus subtilis as described in WO18122225 and an N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34,37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferasepolypeptides identified as described in Example 21. To furtherfucosylate the LNB or LacNAc, the LNB or LacNAc producing strains arefurther modified with a constitutive transcriptional unit for analpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori (GenBankNo. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like, e.g.,HpFucT from H. pylori (UniProt ID 030511).

In an example for the production of lactose-based oligosaccharides, amutant C. glutamicum strain is created to contain a gene coding for alactose importer (such as, e.g., the E. coli lacY with UniProt IDP02920). For 2′FL, 3FL and diFL production, analpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori (GenBankNo. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like, e.g.,HpFucT from H. pylori (UniProt ID 030511) is additionally added to thestrain.

In an example for the production of lacto-N-triose (LNT-II, LN3,GlcNAc-b1,3-Gal-b1,4-Glc), the C. glutamicum strain is modified with agenomic knock-in of constitutive transcriptional units comprising alactose importer (such as, e.g., the E. coli lacY with UniProt IDP02920) and a galactoside beta-1,3-N-acetylglucosaminyltransferase like,e.g., LgtA from N. meningitidis (GenBank: AAM33849.1). For LNTproduction, the LN3 producing strain is further modified with aconstitutive transcriptional unit for an N-acetylglucosaminebeta-1,3-galactosyltransferase chosen from the list comprising SEQ IDNOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDsA0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3,U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8,A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3,A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3,A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2,A0A5C5Y5M7, A0A1G9SAW9, E3H1B28 and A0A538TXM3, that can be delivered tothe strain either via genomic knock-in or from an expression plasmid.For LNnT production, the LN3 producing strain is further modified with aconstitutive transcriptional unit for an N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34,37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferasepolypeptides identified as described in Example 21. To furtherfucosylate the LN3, LNT or LNnT, the mutant strains are further modifiedwith a constitutive transcriptional unit for analpha-1,2-fucosyltransferase like, e.g., HpFutC from H. pylori (GenBankNo. AAD29863.1) and/or an alpha-1,3-fucosyltransferase like, e.g.,HpFucT from H. pylori (UniProt ID 030511).

In an example for sialic acid production, a mutant C. glutamicum strainis created by overexpressing the native fructose-6-P-aminotransferase(UniProt ID Q8NND3) to enhance the intracellular glucosamine-6-phosphatepool. Further on, the enzymatic activities of the C. glutamicum genesldh, cg12645, nagB, gamA and nagA are disrupted by genetic knockouts anda glucosamine-6-P-aminotransferase from S. cerevisiae (UniProt IDP43577), an N-acetylglucosamine-2-epimerase from B. ovatus (UniProt IDA7LVG6) and an N-acetylneuraminate synthase from C. jejuni (UniProt IDQ93MP9) are overexpressed on the genome. To allow sialylatedoligosaccharide production, the sialic acid producing strain is furthermodified with expression constructs comprising an N-acylneuraminatecytidylyltransferase like, e.g., the NeuA enzyme from C. jejuni (UniProtID Q93MP7), the NeuA enzyme from H. influenzae (GenBank No. AGV11798.1)or the NeuA enzyme from P. multocida (GenBank No. AMK07891.1), and oneor more copies of a beta-galactoside alpha-2,3-sialyltransferase like,e.g., PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-likepolypeptide consisting of amino acid residues 1 to 268 of UniProt IDQ9CLP3 having beta-galactoside alpha-2,3-sialyltransferase activity,NmeniST3 from N. meningitidis (GenBank No. ARC07984.1) or PmultST2 fromP. multocida subsp. multocida str. Pm70 (GenBank No. AAK02592.1), abeta-galactoside alpha-2,6-sialyltransferase like, e.g., PdST6 fromPhotobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptideconsisting of amino acid residues 108 to 497 of UniProt ID 066375 havingbeta-galactoside alpha-2,6-sialyltransferase activity orP-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1)or a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues18 to 514 of UniProt ID A8QYL1 having beta-galactosidealpha-2,6-sialyltransferase activity, and/or analpha-2,8-sialyltransferase like, e.g., from M. musculus (UniProt IDQ64689). In an example for the production of lactose-based sialylatedoligosaccharides, the C. glutamicum mutant strains are further modifiedwith a constitutive transcriptional unit for a lactose importer (suchas, e.g., the E. coli lacY with UniProt ID P02920).

For growth on sucrose, the mutant strains can additionally be modifiedwith genomic knock-ins of constitutive transcriptional units comprisingthe sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), thefructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and thesucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6).

Heterologous and homologous expression: Genes that needed to beexpressed, be it from a plasmid or from the genome were syntheticallysynthetized with one of the following companies: DNA2.0, Gen9, TwistBiosciences or IDT.

Expression could be further facilitated by optimizing the codon usage tothe codon usage of the expression host. Genes were optimized using thetools of the supplier.

Cultivation conditions: A pre-culture of 96-well microtiter plateexperiments was started from a cryovial or a single colony from a TYplate, in 150 μL TY and was incubated overnight at 37° C. on an orbitalshaker at 800 rpm. This culture was used as inoculum for a 96-wellsquare microtiter plate, with 400 μL MMsf medium by diluting 400×. Eachstrain was grown in multiple wells of the 96-well plate as biologicalreplicates. These final 96-well culture plates were then incubated at37° C. on an orbital shaker at 800 rpm for 72 hours, or shorter, orlonger. At the end of the cultivation experiment samples were taken fromeach well to measure the supernatant concentration (extracellular sugarconcentrations, after 5 min. spinning down the cells), or by boiling theculture broth for 15 min. at 60° C. before spinning down the cells(=whole broth concentration, intra- and extracellular sugarconcentrations, as defined herein).

Also, a dilution of the cultures was made to measure the optical densityat 600 nm. The cell performance index or CPI was determined by dividingthe oligosaccharide concentrations measured in the whole broth by thebiomass, in relative percentages compared to the reference strain. Thebiomass is empirically determined to be approximately 1/3rd of theoptical density measured at 600 nm.

Example 39: Production of 2′FLNB with a Modified C. glutamicum Strain

A C. glutamicum strain is first modified for LNB production and growthon sucrose by genomic knock-out of the ldh, cg12645 and nagB genes andgenomic knock-ins of constitutive transcriptional units comprising genesencoding the mutant glmS*54 from E. coli (differing from the wild-typeE. coli glmS, having UniProt ID P17169, by an A39T, an R250C and a G472Smutation as described by Deng et a1. (Biochimie 88, 419-29 (2006)), theglucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae(UniProt ID P43577), the phosphatase AraL from B. subtilis (UniProt IDP94526), the N-acetylglucosamine beta-1,3-galactosyltransferase chosenfrom the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and thepolypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10,A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3,A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259,A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1,A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2,F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 andA0A538TXM3, the sucrose transporter (CscB) from E. coli W (UniProt IDEOIXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417)and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt IDA0ZZH6). In a next step, the LNB producing strain is transformed with anexpression plasmid comprising a constitutive transcriptional unit forthe alpha-1,2-fucosyltransferase HpFutC from H. pylori (GenBank No.AAD29863.1). The novel strain is evaluated for the production 2′FLNB ina growth experiment on MMsf medium lacking a precursor according to theculture conditions provided in Example 38. After 72 hours of incubation,the culture broth is harvested, and the sugars are analyzed on UPLC.

Example 40: Production of a Mixture Comprising Sialylated LacNAc with aModified C. glutamicum Strain

A C. glutamicum strain is first modified for LacNAc production andgrowth on sucrose by genomic knock-out of the ldh, cgl2645, nagB, nagAand gamA genes and genomic knock-ins of constitutive transcriptionalunits comprising genes encoding the native fructose-6-P-aminotransferase(UniProt ID Q8NND3), the mutant glmS*54 from E. coli (differing from thewild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250Cand a G472S mutation as described by Deng et a1. (Biochimie 88, 419-29(2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from S.cerevisiae (UniProt ID P43577), the phosphatase AraL from B. subtilis(UniProt ID P94526), the N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34,37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferasepolypeptides identified as described in Example 21, the sucrosetransporter (CscB) from E. coli W (UniProt ID EOIXR1), the fructosekinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrosephosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6). In a nextstep for sialic acid synthesis, the mutant strain was further modifiedwith genomic knock-ins of constitutive transcriptional units comprisinggenes encoding the N-acetylglucosamine-2-epimerase from B. ovatus(UniProt ID A7LVG6), and the N-acetylneuraminate synthase from C. jejuni(UniProt ID Q93MP9). In a next step, the novel strain is transformedwith an expression plasmid comprising constitutive transcriptional unitscomprising the gene encoding the NeuA enzyme from C. jejuni (UniProt IDQ93MP7) combined with the gene encoding either the beta-galactosidealpha-2,3-sialyltransferase PmultST3 from P. multocida (UniProt IDQ9CLP3) or the beta-galactoside alpha-2,6-sialyltransferase PdST6 fromP. damselae (UniProt ID 066375). The novel strains are evaluated forproduction of LacNAc, sialic acid and sialylated LacNAc in a growthexperiment on MMsf medium lacking a precursor according to the cultureconditions provided in Example 38. When adding lactose as precursor tothe MMsf medium, the mutant strains are also evaluated for additionalproduction of 3′-SL or 6′-SL, depending on the alpha-sialyltransferaseexpressed. After 72 hours of incubation, the culture broth is harvested,and the sugars are analyzed on UPLC.

Example 41: Production of Gal-b1,4-GalNAc with a Modified C. glutamicumStrain

A C. glutamicum strain is modified with a genomic knock-in of aconstitutive expression construct for the UDP-glucose4-epimerase (galE)of E. coli (UniProt ID P09147), an N-acetylglucosamineβ1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37,38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferasepolypeptides identified as described in Example 21, the sucrosetransporter CscB from E. coli W (UniProt ID EOIXR1), the fructose kinaseFrk originating from Z. mobilis (UniProt ID Q03417) and the sucrosephosphorylase BaSP from B. adolescentis (UniProt ID A0ZZH6). The novelstrain is evaluated for the production of Gal-b1,4-GalNAc whencultivated in a growth experiment according to the culture conditionsprovided in Example 38, in which the culture medium contains sucrose andGalNAc.

Example 42: Production of Gal-b1,3-GalNAc with a Modified C. glutamicumStrain

A C. glutamicum strain is modified with a genomic knock-in of aconstitutive expression construct for the UDP-glucose4-epimerase (galE)of E. coli (UniProt ID P09147), an N-acetylglucosamineβ1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs:3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93,A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4,T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0,A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0,A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8,A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7,A0A1G9SAW9, E3HB28 and A0A538TXM3, the sucrose transporter CscB from E.coli W (UniProt ID E0IXR1), the fructose kinase Frk originating from Z.mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B.adolescentis (UniProt ID A0ZZH6). The novel strain is evaluated for theproduction of Gal-b1,3-GalNAc when cultivated in a growth experimentaccording to the culture conditions provided in Example 38, in which theculture medium contains sucrose and GalNAc.

Example 43: Materials and Methods Chlamydomonas reinhardtii

Media: C. reinhardtii cells were cultured in Tris-acetate-phosphate(TAP) medium (pH 7.0). The TAP medium uses a 1000× stock Hutner's traceelement mix. Hutner's trace element mix consisted of 50 g/L Na2EDTA·H2O(Titriplex III), 22 g/L ZnSO4.7H2O, 11.4 g/L H3BO3, 5 g/L MnCl2·4H2O, 5g/L FeSO4.7H2O, 1.6 g/L CoCl2·6H2O, 1.6 g/L CuSO4·5H2O and 1.1 g/L(NH4)6MoO3.

The TAP medium contained 2.42 g/L Tris(tris(hydroxymethyl)aminomethane), 25 mg/L salt stock solution, 0.108g/L K2HPO4, 0.054 g/L KH2PO4 and 1.0 mL/L glacial acetic acid. The saltstock solution consisted of 15 g/L NH4CL, 4 g/L MgSO4.7H₂O and 2 g/LCaCl2·2H2O. As precursor for saccharide synthesis, precursors like,e.g., galactose, glucose, fructose and/or fucose could be added. Mediumwas sterilized by autoclaving (121° C., 21 min.). For stock cultures onagar slants TAP medium was used containing 1% agar (of purified highstrength, 1000 g/cm²).

Strains, plasmids and mutations: C. reinhardtii wild-type strains 21 gr(CC-1690, wild-type, mt+), 6145C (CC-1691, wild-type, mt−), CC-125(137c, wild-type, mt+), CC-124 (137c, wild-type, mt−) as available fromChlamydomonas Resource Center (chlamycollection.org), University ofMinnesota, U.S.A.

Expression plasmids originated from pSI103, as available fromChlamydomonas Resource Center. Cloning can be performed using GibsonAssembly, Golden Gate assembly, Cliva assembly, LCR or restrictionligation. Suitable promoters for (heterologous) gene expression can bederived from, e.g., Scranton et a1. (Algal Res. 2016, 15: 135-142).Targeted gene modification (like gene knock-out or gene replacement) canbe carried using the Crispr-Cas technology as described, e.g., by Jianget a1. (Eukaryotic Cell 2014, 13(11): 1465-1469).

Transformation via electroporation was performed as described by Wang eta1. (Biosci. Rep. 2019, 39: BSR2018210). Cells were grown in liquid TAPmedium under constant aeration and continuous light with a lightintensity of 8000 Lx until the cell density reached 1.0−2.0×107cells/mL. Then, the cells were inoculated into fresh liquid TAP mediumin a concentration of 1.0×106 cells/mL and grown under continuous lightfor 18-20 hours until the cell density reached 4.0×106 cells/mL. Next,cells were collected by centrifugation at 1250 g for 5 min. at roomtemperature, washed and resuspended with pre-chilled liquid TAP mediumcontaining 60 mM sorbitol (Sigma, U.S.A.), and iced for 10 min. Then,250 μL of cell suspension (corresponding to 5.0×107 cells) were placedinto a pre-chilled 0.4 cm electroporation cuvette with 100 ng plasmidDNA (400 ng/mL). Electroporation was performed with 6 pulses of 500 Veach having a pulse length of 4 ms and pulse interval time of 100 msusing a BTX ECM830 electroporation apparatus (1575Ω, 50 μFD). Afterelectroporation, the cuvette was immediately placed on ice for 10 min.Finally, the cell suspension was transferred into a 50 ml conicalcentrifuge tube containing 10 mL of fresh liquid TAP medium with 60 mMsorbitol for overnight recovery at dim light by slowly shaking. Afterovernight recovery, cells were recollected and plated with starchembedding method onto selective 1.5% (w/v) agar-TAP plates containingampicillin (100 mg/L) or chloramphenicol (100 mg/L). Plates were thenincubated at 23+−0.5° C. under continuous illumination with a lightintensity of 8000 Lx. Cells were analyzed 5-7 days later.

In an example for production of UDP-galactose, C. reinhardtii cells aremodified with transcriptional units comprising the gene encoding thegalactokinase from Arabidopsis thaliana (KIN, UniProt ID Q9SEE5) and thegene encoding the UDP-sugar pyrophosphorylase (USP) from A. thaliana(UniProt ID Q9C5I1).

In an example for production of LNB, C. reinhardtii cells modified forUDP-galactose production are further modified with an expression plasmidcomprising a transcriptional unit for the N-acetylglucosaminebeta-1,3-galactosyltransferase chosen from the list comprising SEQ IDNOs: 3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDsA0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3,U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8,A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3,A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3,A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2,A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3. Additionally, the mutantC. reinhardtii cells can be modified with an expression plasmidcomprising a transcriptional unit for an alpha-1,2-fucosyltransferaselike, e.g., HpFutC from H. pylori (GenBank No. AAD29863.1) and/or analpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProtID 030511).

In an example for production of LacNAc, C. reinhardtii cells modifiedfor UDP-galactose production are further modified with an expressionplasmid comprising a transcriptional unit for the N-acetylglucosaminebeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34,37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferasepolypeptides identified as described in Example 21. Additionally, themutant C. reinhardtii cells can be modified with an expression plasmidcomprising a transcriptional unit for an alpha-1,2-fucosyltransferaselike, e.g., HpFutC from H. pylori (GenBank No. AAD29863.1) and/or analpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProtID 030511).

In an example for CMP-sialic acid synthesis, C. reinhardtii cells aremodified with constitutive transcriptional units for aUDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase like,e.g., GNE from Homo sapiens (UniProt ID Q9Y223) or a mutant form of thehuman GNE polypeptide comprising the R263L mutation, anN-acylneuraminate-9-phosphate synthetase like, e.g., NANS from Homosapiens (UniProt ID Q9NR45) and an N-acylneuraminatecytidylyltransferase like, e.g., CMAS from Homo sapiens (UniProt IDQ8NFW8). In an example for production of sialylated oligosaccharides, C.reinhardtii cells are modified with a CMP-sialic acid transporter like,e.g., CST from Mus musculus (UniProt ID Q61420), and a Golgi-localisedsialyltransferase chosen from species like, e.g., Homo sapiens, Musmusculus, and Rattus norvegicus.

Heterologous and homologous expression: Genes that needed to beexpressed, be it from a plasmid or from the genome were syntheticallysynthetized with one of the following companies: DNA2.0, Gen9, TwistBiosciences or IDT.

Expression could be further facilitated by optimizing the codon usage tothe codon usage of the expression host. Genes were optimized using thetools of the supplier.

Cultivation conditions: Cells of C. reinhardtii were cultured inselective TAP-agar plates at 23+/−0.5° C. under 14/10-hour light/darkcycles with a light intensity of 8000 Lx. Cells were analyzed after 5 to7 days of cultivation.

For high-density cultures, cells could be cultivated in closed systemslike, e.g., vertical or horizontal tube photobioreactors, stirred tankphotobioreactors or flat panel photobioreactors as described by Chen eta1. (Bioresour. Technol. 2011, 102: 71-81) and Johnson et a1.(Biotechnol. Prog. 2018, 34: 811-827).

Example 44: Production of LNB and 2′FLNB in Modified C. reinhardtiiCells

C. reinhardtii cells are engineered as described in Example 43 forproduction of UDP-Gal with genomic knock-ins of constitutivetranscriptional units comprising the galactokinase from A. thaliana(KIN, UniProt ID Q9SEE5) and the UDP-sugar pyrophosphorylase (USP) fromA. thaliana (UniProt ID Q9C5I1). In a next step, the mutant cells aretransformed with an expression plasmid comprising transcriptional unitscomprising an N-acetylglucosamine beta-1,3-galactosyltransferase chosenfrom the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and thepolypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10,A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3,A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259,A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1,A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2,F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 andA0A538TXM3 and the alpha-1,2-fucosyltransferase HpFutC from H. pylori(GenBank No. AAD29863.1). The novel strain is evaluated in a cultivationexperiment on TAP-agar plates comprising galactose as precursoraccording to the culture conditions provided in Example 43. After 5 daysof incubation, the cells are harvested, and evaluated for the productionof LNB and 2′FLNB via analysis on UPLC.

Example 45: Production of LacNAc and 3′-Fucosylated LacNAc (3-FLN) inModified C. reinhardtii Cells

C. reinhardtii cells are engineered as described in Example 43 forproduction of UDP-Gal with genomic knock-ins of constitutivetranscriptional units comprising the galactokinase from A. thaliana(KIN, UniProt ID Q9SEE5) and the UDP-sugar pyrophosphorylase (USP) fromA. thaliana (UniProt ID Q9C5I1). In a next step, the mutant cells aretransformed with an expression plasmid comprising transcriptional unitscomprising an N-acetylglucosamine beta-1,4-galactosyltransferase chosenfrom the list comprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23,26, 27, 29, 30, 31, 32, 33, 34, 37, 38 and 39 and theN-acetylglucosamine b-1,4-galactosyltransferase polypeptides and thealpha-1,3-fucosyltransferase like, e.g., HpFucT from H. pylori (UniProtID 030511). The novel strain is evaluated in a cultivation experiment onTAP-agar plates comprising galactose as precursor according to theculture conditions provided in Example 43. After 5 days of incubation,the cells are harvested, and cells are evaluated for the production ofLacNAc and 3′-fucosylated LacNAc (3-FLN, Gal-b1,4-(Fuc-a1,3)-GlcNAc) viaanalysis on UPLC.

Example 46: Production of Gal-b1,4-GalNAc with a Modified C. reinhardtiiStrain

C. reinhardtii cells are modified with a genomic knock-in of aconstitutive expression construct for the UDP-glucose4-epimerase (galE)of E. coli (UniProt ID P09147), and an N-acetylglucosamineβ1,4-galactosyltransferase chosen from the list comprising SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 37,38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferasepolypeptides identified as described in Example 21. The novel strain isevaluated in a cultivation experiment on TAP-agar plates comprisingGalNAc as precursor according to the culture conditions provided inExample 43. After 5 days of incubation, the cells are harvested, andcells are evaluated for the production of Gal-b1,4-GalNAc via analysison UPLC.

Example 47: Production of Gal-b1,3-GalNAc with a Modified C. reinhardtiiStrain

C. reinhardtii cells are modified with a genomic knock-in of aconstitutive expression construct for the UDP-glucose4-epimerase (galE)of E. coli (UniProt ID P09147) and an N-acetylglucosamineβ1,3-galactosyltransferase chosen from the list comprising SEQ ID NOs:3, 4, 6, 7, 8 and 9 and the polypeptides with UniProt IDs A0A354SD93,A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4,T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0,A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0,A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8,A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7,A0A1G9SAW9, E3HB28 and A0A538TXM3. The novel strain is evaluated in acultivation experiment on TAP-agar plates comprising GalNAc as precursoraccording to the culture conditions provided in Example 43. After 5 daysof incubation, the cells are harvested, and cells are evaluated for theproduction of Gal-b1,3-GalNAc via analysis on UPLC.

Example 48: Materials and Methods Animal Cells

Isolation of mesenchymal stem cells from adipose tissue of differentmammals: Fresh adipose tissue is obtained from slaughterhouses (e.g.,cattle, pigs, sheep, chicken, ducks, catfish, snake, frogs) orliposuction (e.g., in case of humans, after informed consent) and keptin phosphate buffer saline supplemented with antibiotics. Enzymaticdigestion of the adipose tissue is performed followed by centrifugationto isolate mesenchymal stem cells. The isolated mesenchymal stem cellsare transferred to cell culture flasks and grown under standard growthconditions, e.g., 37° C., 5% CO2. The initial culture medium includesDMEM-F12, RPMI, and Alpha-MEM medium (supplemented with 15% fetal bovineserum), and 1% antibiotics. The culture medium is subsequently replacedwith 10% FBS (fetal bovine serum)-supplemented media after the firstpassage. For example, Ahmad and Shakoori (2013, Stem Cell Regen. Med.9(2): 29-36), which is incorporated herein by reference in its entiretyfor all purposes, describes certain variation(s) of the method(s)described herein in this example.

Isolation of mesenchymal stem cells from milk: This example illustratesisolation of mesenchymal stem cells from milk collected under asepticconditions from human or any other mammal(s) such as described herein.An equal volume of phosphate buffer saline is added to diluted milk,followed by centrifugation for 20 min. The cell pellet is washed thricewith phosphate buffer saline and cells are seeded in cell culture flasksin DMEM-F12, RPMI, and Alpha-MEM medium supplemented with 10% fetalbovine serum and 1% antibiotics under standard culture conditions. Forexample, Hassiotou et a1. (2012, Stem Cells. 30(10): 2164-2174), whichis incorporated herein by reference in its entirety for all purposes,describes certain variation(s) of the method(s) described herein in thisexample.

Differentiation of stem cells using 2D and 3D culture systems: Theisolated mesenchymal cells can be differentiated into mammary-likeepithelial and luminal cells in 2D and 3D culture systems. See, forexample, Huynh et al. 1991. Exp. Cell Res. 197(2): 191-199; Gibson etal. 1991, In vitro Cell Dev. Biol. Anim. 27(7): 585-594; Blatchford etal. 1999; Animal Cell Technology: Basic & Applied Aspects, Springer,Dordrecht. 141-145; Williams et al. 2009, Breast Cancer Res. 11(3):26-43; and Arevalo et al. 2015, Am. J. Physiol. Cell Physiol. 310(5):C348-C356; each of which is incorporated herein by reference in theirentireties for all purposes.

For 2D culture, the isolated cells were initially seeded in cultureplates in growth media supplemented with 10 ng/ml epithelial growthfactor and 5 pg/ml insulin. At confluence, cells were fed with growthmedium supplemented with 2% fetal bovine serum, 1%penicillin-streptomycin (100 U/ml penicillin, 100 ug/ml streptomycin),and 5 pg/ml insulin for 48 hours. To induce differentiation, the cellswere fed with complete growth medium containing 5 pg/ml insulin, 1 pg/mlhydrocortisone, 0.65 ng/ml triiodothyronine, 100 nM dexamethasone, and 1pg/ml prolactin. After 24 hours, serum is removed from the completeinduction medium.

For 3D culture, the isolated cells were trypsinized and cultured inMatrigel, hyaluronic acid, or ultra-low attachment surface cultureplates for six days and induced to differentiate and lactate by addinggrowth media supplemented with 10 ng/ml epithelial growth factor and 5pg/ml insulin. At confluence, cells were fed with growth mediumsupplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100U/ml penicillin, 100 ug/ml streptomycin), and 5 pg/ml insulin for 48hours. To induce differentiation, the cells were fed with completegrowth medium containing 5 pg/ml insulin, 1 pg/ml hydrocortisone, 0.65ng/ml triiodothyronine, 100 nM dexamethasone, and 1 pg/ml prolactin.After 24 hours, serum is removed from the complete induction medium.

Method of making mammary-like cells: Mammalian cells are brought toinduced pluripotency by reprogramming with viral vectors encoding forOct4, Sox2, Klf4, and c-Myc. The resultant reprogrammed cells are thencultured in Mammocult media (available from Stem Cell Technologies), ormammary cell enrichment media (DMEM, 3% FBS, estrogen, progesterone,heparin, hydrocortisone, insulin, EGF) to make them mammary-like, fromwhich expression of select milk components can be induced.Alternatively, epigenetic remodeling are performed using remodelingsystems such as CRISPR/Cas9, to activate select genes of interest, suchas casein, a-lactalbumin to be constitutively on, to allow for theexpression of their respective proteins, and/or to down-regulate and/orknock-out select endogenous genes as described, e.g., in WO 2021067641,which is incorporated herein by reference in its entirety for allpurposes.

Cultivation: Completed growth media includes high glucose DMEM/F12, 10%FBS, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF, and 5pg/ml hydrocortisone. Completed lactation media includes high glucoseDMEM/F12, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF, 5pg/ml hydrocortisone, and 1 pg/ml prolactin (5 ug/ml in Hyunh 1991).Cells are seeded at a density of 20,000 cells/cm² onto collagen coatedflasks in completed growth media and left to adhere and expand for 48hours in completed growth media, after which the media is switched outfor completed lactation media. Upon exposure to the lactation media, thecells start to differentiate and stop growing. Within about a week, thecells start secreting lactation product(s) such as milk lipids, lactose,casein and whey into the media. A desired concentration of the lactationmedia can be achieved by concentration or dilution by ultrafiltration. Adesired salt balance of the lactation media can be achieved by dialysis,for example, to remove unwanted metabolic products from the media.Hormones and other growth factors used can be selectively extracted byresin purification, for example, the use of nickel resins to removeHis-tagged growth factors, to further reduce the levels of contaminantsin the lactated product.

Example 49: Evaluation of 2′FL, LNFP-I and 2′FLNB Production in aNon-Mammary Adult Stem Cell

Isolated mesenchymal cells and re-programmed into mammary-like cells asdescribed in Example 48 are modified via CRISPR-CAS to over-express acodon-optimized N-acetylglucosamine beta-1,3-galactosyltransferasechosen from the list comprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and thepolypeptides with UniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10,A0A1G2UER6, A0A1G2UVR8, T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3,A0A0M7B6J0, A0A0K1Q500, A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259,A0A5C9C3N6, A0A1G7VWF9, A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1,A0A5N1JGF2, A0A538SYW6, N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2,F3PEK1, B0NR63, A0A3D3JDC2, A0A5C5Y5M7, A0A1G9SAW9, E3HB28 andA0A538TXM3, the GDP-fucose synthase GFUS from Homo sapiens (UniProt IDQ13630), and a codon-optimized alpha-1,2-fucosyltransferase from H.pylori (GenBank No. AAD29863.1). Cells are seeded at a density of 20,000cells/cm² onto collagen coated flasks in completed growth media and leftto adhere and expand for 48 hours in completed growth media, after whichthe media is switched out for completed lactation media for about 7days. After cultivation as described in Example 48, cells are subjectedto UPLC to analyze for production of 2′FL, LNFP-I and 2′FLNB.

Example 50: Evaluation of LacNAc, Sialylated LacNAc andSialyl-Lewis×Production in a Non-Mammary Adult Stem Cell

Isolated mesenchymal cells and re-programmed into mammary-like cells asdescribed in Example 48 are modified via CRISPR-CAS to over-express abeta-1,4-galactosyltransferase chosen from the list comprising SEQ IDNOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34,37, 38 and 39 and the N-acetylglucosamine b-1,4-galactosyltransferasepolypeptides identified as described in Example 21, the GDP-fucosesynthase GFUS from Homo sapiens (UniProt ID Q13630), the galactosidealpha-1,3-fucosyltransferase FUT3 from H. sapiens (UniProt ID P21217),the N-acylneuraminate cytidylyltransferase from Mus musculus (UniProt IDQ99KK2) and the CMP-N-acetylneuraminate-beta-1,4-galactosidealpha-2,3-sialyltransferase ST3GAL3 from Homo sapiens (UniProt IDQ11203). All genes introduced in the cells are codon-optimized to thehost. Cells are seeded at a density of 20,000 cells/cm² onto collagencoated flasks in completed growth media and left to adhere and expandfor 48 hours in completed growth media, after which the media isswitched out for completed lactation media for about 7 days. Aftercultivation as described in Example 48, cells are subjected to UPLC toanalyze for production of LacNAc, sialylated LacNAc and sialyl-Lewis x.

Example 51: Evaluation of Production of Gal-b1,4-GalNAc in a Non-MammaryAdult Stem Cell

Isolated mesenchymal cells and re-programmed into mammary-like cells asdescribed in Example 48 are modified via CRISPR-CAS to over-express theUDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147), and anN-acetylglucosamine β1,4-galactosyltransferase chosen from the listcomprising SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29,30, 31, 32, 33, 34, 37, 38 and 39 and the N-acetylglucosamineb-1,4-galactosyltransferase polypeptides identified as described inExample 21. Both genes introduced in the cells are codon-optimized tothe host. Cells are seeded at a density of 20,000 cells/cm² ontocollagen coated flasks in completed growth media and left to adhere andexpand for 48 hours in completed growth media, after which the media isswitched out for completed lactation media for about 7 days. Aftercultivation as described in Example 48, cells are subjected to UPLC toanalyze for production of Gal-b1,4-GalNAc.

Example 52: Evaluation of Production of Gal-b1,3-GalNAc in a Non-MammaryAdult Stem Cell

Isolated mesenchymal cells and re-programmed into mammary-like cells asdescribed in Example 48 are modified via CRISPR-CAS to over-express theUDP-glucose4-epimerase (galE) of E. coli (UniProt ID P09147), and anN-acetylglucosamine β1,4-galactosyltransferase chosen from the listcomprising SEQ ID NOs: 3, 4, 6, 7, 8 and 9 and the polypeptides withUniProt IDs A0A354SD93, A0A108TBL4, A0A1G2TK10, A0A1G2UER6, A0A1G2UVR8,T1RPX3, U4SLB4, T1RNY4, T1RQ38, A0A377HVE3, A0A0M7B6J0, A0A0K1Q500,A0A3S0CAP8, A0A5C8BKY0, A0A0G0R169, A0A5C6Y259, A0A5C9C3N6, A0A1G7VWF9,A0A193KHC3, A0A5N1GML0, A0A3N2I9V8, A0A2I1RGW1, A0A5N1JGF2, A0A538SYW6,N8U0B3, A0A1G8DZV8, A0A538U133, A0A538SYT2, F3PEK1, B0NR63, A0A3D3JDC2,A0A5C5Y5M7, A0A1G9SAW9, E3HB28 and A0A538TXM3. Both genes introduced inthe cells are codon-optimized to the host. Cells are seeded at a densityof 20,000 cells/cm² onto collagen coated flasks in completed growthmedia and left to adhere and expand for 48 hours in completed growthmedia, after which the media is switched out for completed lactationmedia for about 7 days. After cultivation as described in Example 48,cells are subjected to UPLC to analyze for production ofGal-b1,3-GalNAc.

Example 53: Production of LNB in a Modified E. coli Host

An E. coli K-12 MG1655 strain optimized for GDP-fucose production wasmodified with genomic knock-ins of constitutive expression constructsfor glmS*54 (differing from the wild-type glmS protein (UniProt IDP17169) by an A39T, an R250C and a G472S mutation) and GNA1 from S.cerevisiae (UniProt ID P43577) to intracellularly produce GlcNAc. In anext step, the mutant strain was further modified for constitutiveexpression of the N-acetylglucosamine β1,3-galactosyltransferase fromPhotobacterium leiognathi with SEQ ID NO: 4 from plasmid. The novelstrain was evaluated in a growth experiment according to the cultureconditions provided in Example 1. The strain demonstrated that thestrain produced 0.37 (±0.18) g/L LNB in whole broth samples, taken after72 hours of cultivation in minimal medium with 30 g/L sucrose. The cellperformance index (CPI) was determined by dividing the LNB concentrationmeasured in the whole broth by the biomass. The biomass was empiricallydetermined to be approximately ⅓rd of the optical density measured at600 nm. The CPI related to LNB production in whole broth samples for thenovel strain was 0.10±0.05.

Example 54: Production of LacNAc in Modified E. coli Hosts

An E. coli K-12 MG1655 strain optimized for GDP-fucose production wasmodified with genomic knock-ins of constitutive expression constructsfor glmS*54 (differing from the wild-type glmS protein (UniProt IDP17169) by an A39T, an R250C and a G472S mutation) and GNA1 from S.cerevisiae (UniProt ID P43577) to intracellularly produce GlcNAc. In anext step, the mutant strain was further modified for constitutiveexpression of an N-acetylglucosamine β1,4-galactosyltransferase chosenfrom the list comprising SEQ ID NOs: 17, 20, 22, 27 and 37 from plasmid.The novel strains were evaluated in a growth experiment according to theculture conditions provided in Example 1. Table 3 shows the productionof LacNAc (g/L) in whole broth samples from each of the mutant strains,taken after 72 hours of cultivation in minimal medium with 30 g/Lsucrose, together with the cell performance index (CPI) calculated forthe samples. The CPI was determined by dividing the LacNAcconcentrations, measured in the whole broth by the biomass. The biomasswas empirically determined to be approximately ⅓rd of the opticaldensity measured at 600 nm. The data demonstrated that all novel strainsexpressing either SEQ ID NOs: 17, 20, 22, 27 or 37 produced LacNAc.

TABLE 3 Production of LacNAc (g/L) and cell performance index (CPI) datarelated to the LacNAc production in whole broth samples taken frommutant E. coli strains after 72 hours of cultivation in minimal mediumcomprising 30 g/L sucrose SEQ ID NO of the β1,4-galactosyltransferaseexpressed from a transcriptional unit from an LacNAc CPI_ expressionplasmid (g/L) (± sd) LacNAc (± sd) 17  1.89 ± 0.10 0.50 ± 0.01 20  9.32± 4.45 2.55 ± 1.24 22  3.40 ± 0.39 0.93 ± 0.05 27  9.33 ± 0.20 2.51 ±0.13 37 10.50 ± 0.48 2.94 ± 0.11

Example 55: Production of LNT in a Modified E. coli Host

An E. coli K-12 strain MG1655 was modified as described in Example 1comprising knock-outs of the E. coli nagB, galT, ushA, agp, ldhA, LacZ,LacY and LacA genes and genomic knock-ins of constitutivetranscriptional units comprising the genes encoding the lactose permease(LacY) from E. coli (UniProt ID P02920), the sucrose transporter (CscB)from E. coli W (UniProt ID EOIXR1), the fructose kinase (Frk) from Z.mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B.adolescentis (UniProt ID 35 A0ZZH6). In a next step, the mutant E. colistrain was modified for LN3 production with a genomic knock-in of aconstitutive transcriptional unit for the galactosidebeta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis(GenBank: AAM33849.1). In a further step, the mutant strain was modifiedfor LNT production with a constitutive transcriptional unit for anN-acetylglucosamine β1,3-galactosyltransferase expressed from plasmidchosen from the list comprising SEQ ID NOs: 3 and 4. The novel strainswere evaluated in a growth experiment according to the cultureconditions provided in Example 1. Table 4 shows the production of LNT inwhole broth samples from each of the mutant strains, taken after 72hours of cultivation in minimal medium with 30 g/L sucrose, togetherwith the cell performance index (CPI) calculated for the samples. TheCPI was determined by dividing the LNT concentrations, measured in thewhole broth by the biomass. The biomass was empirically determined to beapproximately ⅓rd of the optical density measured at 600 nm. Theexperiment demonstrated that the strains expressing aN-acetylglucosamine β1,3-galactosyltransferase with SEQ ID NOs: 3 or 4were able to produce LNT.

TABLE 4 Production of LNT (g/L) and cell performance index (CPI) datarelated to the LNT production in whole broth samples taken from mutantE. coli strains after 72 hours of cultivation in minimal mediumcomprising 30 g/L sucrose 20 g/L lactose SEQ ID NO of theβ1,3-galactosyltransferase expressed from a transcriptional unit from anLNT CPI_ expression plasmid (g/L) (± sd) LNT ± sd 03 5.25 ± 1.50 1.54 ±0.42 04 0.83 ± 0.26 0.27 ± 0.06

Example 56: Production of LNnT in a Modified E. coli Host

An E. coli K-12 strain MG1655 was modified as described in Example 1comprising knock-outs of the E. coli nagB, galT, ushA, agp, ldhA, LacZ,LacY and LacA genes and genomic knock-ins of constitutivetranscriptional units comprising the genes encoding the lactose permease(LacY) from E. coli (UniProt ID P02920), the sucrose transporter (CscB)from E. coli W (UniProt ID EOIXR1), the fructose kinase (Frk) from Z.mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B.adolescentis (UniProt ID 20 A0ZZH6). In a next step, the mutant E. colistrain was modified for LN3 production with a genomic knock-in of aconstitutive transcriptional unit for the galactosidebeta-1,3-N-acetylglucosaminyltransferase lgtA from N. meningitidis(GenBank: AAM33849.1). In a further step, the mutant strain was modifiedfor LNnT production with a constitutive transcriptional unit for anN-acetylglucosamine β1,4-galactosyltransferase expressed from plasmidchosen from the list comprising SEQ ID NOs: 15, 17, 18, 19, 20, 22, 32and 33. The novel strains were evaluated in a growth experimentaccording to the culture conditions provided in Example 1. Table 5 showsthe production of LNnT in whole broth samples from each of the mutantstrains, taken after 72 hours of cultivation in minimal medium with 30g/L sucrose, together with the cell performance index (CPI) calculatedfor the samples. The CPI was determined by dividing the LNnTconcentrations, measured in the whole broth by the biomass. The biomasswas empirically determined to be approximately ⅓rd of the opticaldensity measured at 600 nm. The experiment demonstrated that the strainsexpressing a N-acetylglucosamine β1,4-galactosyltransferase with SEQ IDNOs: 15, 17, 18, 19, 20, 22, 32 or 33 were able to produce LNnT.

TABLE 5 Production of LNnT (g/L) and cell performance index (CPI) datarelated to the LNnT production in whole broth samples taken from mutantE. coli strains after 72 hours of cultivation in minimal mediumcomprising 30 g/L sucrose 20 g/L lactose SEQ ID NO of theβ1,4-galactosyltransferase expressed from a transcriptional unit from anLNnT CPI_ expression plasmid (g/L) (± sd) LNnT ± sd 15 2.82 ± 0.82 1.57± 0.49 17 0.59 ± 0.02 0.41 ± 0.01 18 1.64 ± 0.05 0.73 ± 0.06 19 0.58 ±0.04 0.31 ± 0.02 20 0.88 ± 0.16 0.94 ± 0.19 22 1.11 ± 0.13 1.00 ± 0.0233 0.25 ± 0.03 0.24 ± 0.07

What is claimed is:
 1. An N-acetylglucosamineb-1,X-galactosyltransferase that galactosylates an N-acetylglucosamineand/or N-acetylgalactosamine as a monosaccharide, and/or anN-acetylglucosamine and/or N-acetylgalactosamine as part of a di- and/oroligosaccharide at the non-reducing end of the di- and/oroligosaccharide, and wherein the N-acetylglucosamineb-1,X-galactosyltransferase is: A. an N-acetylglucosamineb-1,3-galactosyltransferase which has a. PFAM domain PF00535 and i)comprises the sequence [AGPS]XXLN(X_(n))RXDXD with SEQ ID NO: 1, whereinX is any amino acid except for the combination XX on positions 2 and 3that cannot be an FA, FS, YC or YS combination and wherein n is 12 to 17ii) comprises the sequence PXXLN(X_(n))RXDXD(X_(m))[FWY]XX[HKR]XX[NQST]with SEQ ID NO: 2, wherein X is any amino acid except for thecombination XX on positions 2 and 3 that cannot be an FA, FS, YC or YScombination and wherein n is 12 to 17 and m is 100 to 115, iii)comprises a polypeptide sequence of SEQ ID NO: 3 or 4, or iv) is afunctional homologue, variant or derivative of SEQ ID NO: 3 or 4 havingat least 80% overall sequence identity to the full length of any one ofthe N-acetylglucosamine b-1,3-galactosyltransferase polypeptide with SEQID NOs: 3 or 4 and having N-acetylglucosamineb-1,3-galactosyltransferase activity, v) comprises an oligopeptidesequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20consecutive amino acid residues from SEQ ID NO: 3 or 4 and havingN-acetylglucosamine b-1,3-galactosyltransferase activity, vi) is afunctional fragment of SEQ ID NO: 3 or 4 and having N-acetylglucosamineb-1,3-galactosyltransferase activity, or vii) comprises a polypeptidecomprising a peptide having at least 80% sequence identity to thefull-length peptide of SEQ ID NO: 3 or 4 and having N-acetylglucosamineb-1,3-galactosyltransferase activity, or b. PFAM domain IPR002659 and i)comprises the sequence KT(Xn)[FY]XXKXDXD(Xm)[FHY]XXG(X, no A, G,S)(Xp)(X, no F, H, W, Y)[DE]D[ILV]XX[AG] with SEQ ID NO: 5, wherein X isany amino acid and wherein n is 13 to 16, m is 35 to 70 and p is 20 to45, ii) comprises the polypeptide sequence of any one of SEQ ID NOs: 6,7, 8 or 9, iii) is a functional homologue, variant or derivative of anyone of SEQ ID NOs: 6, 7, 8 or 9 having at least 80% overall sequenceidentity to the full length of any one of the N-acetylglucosamineb-1,3-galactosyltransferase polypeptide with SEQ ID NOs: 6, 7, 8 or 9and having N-acetylglucosamine b-1,3-galactosyltransferase activity, iv)comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one ofSEQ ID NOs: 6, 7, 8, or 9 and having N-acetylglucosamineb-1,3-galactosyltransferase activity, v) is a functional fragment of anyone of SEQ ID NOs: 6, 7, 8, or 9 and having N-acetylglucosamineb-1,3-galactosyltransferase activity, or vi) comprises a polypeptidecomprising a peptide having at least 80% sequence identity to thefull-length peptide of any one of SEQ ID NOs: 6, 7, 8, or 9 and havingN-acetylglucosamine b-1,3-galactosyltransferase activity, or B. anN-acetylglucosamine b-1,4-galactosyltransferase which has a. PFAM domainPF01755 and i) comprises the sequenceEXXCXXSHX[AFILTY]LW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 10,wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75,or ii) comprises the sequence EXXCXXSH[LR]VLW(Xn)EDD(Xm)[ACGST]XXY[ILMV]with SEQ ID NO: 11, wherein X is any amino acid and wherein n is 13 to15 and m is 50 to 75, iii) comprises the sequenceEXXCXXSH[VHI]SLW(X_(n))EDD(X_(m))[ACGST]XXY[ILMV] with SEQ ID NO: 12,wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75,iv) comprises the sequence EXXCXXSHYMLW(X_(n))EDD(Xm)[ACGST]XXY[ILMV]with SEQ ID NO: 13, wherein X is any amino acid and wherein n is 13 to15 and m is 50 to 75, v) comprises the sequence EXXCXXSHXX(X, noV)Y(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 14, wherein X is anyamino acid and wherein n is 13 to 15 and m is 50 to 75, vi) comprisesthe polypeptide sequence of any one of SEQ ID NOs: 15, 18, 22, 20, 17,19, 16, 21 or 23, vii) is a functional homologue, variant or derivativeof any one of SEQ ID NOs: 15, 18, 22, 20, 17, 19, 16, 21 or 23 having atleast 80% overall sequence identity to the full length of any one of theN-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ IDNOs: 15, 18, 22, 20, 17, 19, 16, 21 or 23 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, viii) comprises an oligopeptidesequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20consecutive amino acid residues from any one of SEQ ID NOs: 15, 18, 22,20, 17, 19, 16, 21 or 23 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, ix) is a functional fragment ofany one of SEQ ID NOs: 15, 18, 22, 20, 17, 19, 16, 21 or 23 and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, or x)comprises a polypeptide comprising a peptide having at least 80%sequence identity to the full-length peptide of any one of SEQ ID NOs:15, 18, 22, 20, 17, 19, 16, 21 or 23 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or b. PFAM domain PF00535 and i)comprises the sequence R[KN]XXXXXXXGXXXX[FL](X, noV)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE] with SEQ ID NO: 24 wherein X is anyamino acid and wherein n is 50 to 75 and m is 10 to 30, ii) comprisesthe sequence R[KN]XXXXXXXGXXXX[FL](X, noV)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE](Xp)[FWY]XX[HKR]XX[NQST] with SEQ ID NO:25 wherein X is any amino acid and wherein n is 50 to 75, m is 10 to 30and p is 20 to 25, iii) comprises a polypeptide sequence of SEQ ID NO:26 or 27, iv) is a functional homologue, variant or derivative of SEQ IDNO: 26 or 27 having at least 80% overall sequence identity to the fulllength of any one of the N-acetylglucosamine b-1,4-galactosyltransferasepolypeptide with SEQ ID NO: 26 or 27 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, v) comprises an oligopeptidesequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20consecutive amino acid residues from SEQ ID NO: 26 or 27 and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, vi) is afunctional fragment of SEQ ID NO: 26 or 27 and havingN-acetylglucosamine b-1,4-galactosyltransferase activity, or vii)comprises a polypeptide comprising a peptide having at least 80%sequence identity to the full-length peptide of SEQ ID NO: 26 or 27 andhaving N-acetylglucosamine b-1,4-galactosyltransferase activity, or c.PFAM domain PF02709 and not PFAM domain PF00535, and i) comprises thesequence [FWY]XX[FWY](X_(n))[FWY][GQ]X[DE]D with SEQ ID NO: 28 wherein Xis any amino acid except for the combination XX on positions 2 and 3that cannot be an I1P or NL combination and wherein n is 21 to 26, ii)comprises the polypeptide sequence of any one of SEQ ID NOs: 33, 29, 30,31, 32 or 34, or iii) is a functional homologue, variant or derivativeof any one of SEQ ID NOs: 33, 29, 30, 31, 32 or 34 having at least 80%overall sequence identity to the full length of any one of theN-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ IDNOs: 33, 29, 30, 31, 32 or 34 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, iv) comprises an oligopeptidesequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20consecutive amino acid residues from any one of SEQ ID NOs: 33, 29, 30,31, 32 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferaseactivity, v) is a functional fragment of any one of SEQ ID NOs: 33, 29,30, 31, 32 or 34 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or vi) comprises a polypeptidecomprising a peptide having at least 80% sequence identity to thefull-length peptide of any one of SEQ ID NOs: 33, 29, 30, 31, 32 or 34and having N-acetylglucosamine b-1,4-galactosyltransferase activity, ord. PFAM domain PF03808 and i) comprises the sequence[ST][FHY]XN(Xn)DGXXXXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA with SEQ ID NO:35, wherein X is any amino acid and wherein n is 20 to 25, ii) comprisesthe sequence [ST][FHY]XN(Xn)DGXXXXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA(Xm)[HR]XG[FWY](Xp)GXGXXXQ[DE] with SEQ ID NO: 36, wherein X is anyamino acid and wherein n is 20 to 25, m is 40 to 50 and p is 22 to 30,iii) comprises the polypeptide sequence of any one of SEQ ID NOs: 37, 38or 39, iv) is a functional homologue, variant or derivative of any oneof SEQ ID NOs: 37, 38 or 39 having at least 80% overall sequenceidentity to the full length of any one of the N-acetylglucosamineb-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 37, 38 or 39and having N-acetylglucosamine b-1,4-galactosyltransferase activity, v)comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one ofSEQ ID NOs: 37, 38 or 39 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, vi) functional fragment of any oneof SEQ ID NOs: 37, 38 or 39 and having N-acetylglucosamineb-1,4-galactosyltransferase activity, or vii) comprises a polypeptidecomprising a peptide having at least 80% sequence identity to thefull-length peptide of any one of SEQ ID NOs: 37, 38, or 39 and havingN-acetylglucosamine b-1,4-galactosyltransferase activity.
 2. A method ofsynthesizing a galactosylated disaccharide or oligosaccharide, themethod comprising: utilizing the N-acetylglucosamineb-1,X-galactosyltransferase of claim 1 in a method of synthesizing agalactosylated disaccharide or oligosaccharide.
 3. The method accordingto claim 2, wherein the synthesis comprises: a. providing UDP-galactoseand any one of the galactosyltransferase, wherein thegalactosyltransferase is capable of transferring a galactose residuefrom the UDP-galactose donor to one or more acceptor(s), and b.contacting any one of the galactosyltransferase and UDP-galactose withone or more acceptor(s), under conditions where thegalactosyltransferase catalyses the transfer of a galactose residue fromthe UDP-galactose to the acceptor(s), and c. optionally, separating thegalactosylated di- or oligosaccharide.
 4. The method according to claim3, wherein the acceptor(s) is/are an N-acetylglucosamine and/or anN-acetylgalactosamine as a monosaccharide, and/or a di- and/oroligosaccharide having an N-acetylglucosamine and/orN-acetylgalactosamine at its non-reducing end.
 5. The method accordingto claim 2, wherein the galactosylated disaccharide or oligosaccharideis produced in a cell-free system or is produced by a cell.
 6. Themethod according to claim 5, wherein the galactosylated disaccharide oroligosaccharide is produced by a cell and the cell is capable ofsynthesizing one or more of the acceptor(s), expresses any one of theN-acetylglucosamine b-1,3-galactosyltransferases and/orN-acetylglucosamine b-1,4-galactosyltransferases, and is capable ofsynthesizing UDP-galactose (UDP-Gal) as donor for thegalactosyltransferases.
 7. The method according to claim 5, wherein thegalactosylated disaccharide or oligosaccharide is produced by a cell andthe cell is capable of synthesizing one or more nucleotide-sugardonor(s) selected from the group consisting of GDP-Fuc, CMP-Neu5Ac,UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc),UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose(UDP-Glc), UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc orUDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,UDP-N-acetylfucosamine (UDP-L-FucNAc orUDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamnine(UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose),UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc orUDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose,CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N3,CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2,UDP-glucuronate, UDP-galacturonate, DP-rhamnose, and UDP-xylose.
 8. Themethod according to claim 5, wherein the galactosylated disaccharide oroligosaccharide is produced by a cell and the cell is capable ofexpressing one or more glycosyltransferases selected from the groupconsisting of fucosyltransferases, sialyltransferases,galactosyltransferases, glucosyltransferases, mannosyltransferases,N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases,N-acetylmannosaminyltransferases, xylosyltransferases,glucuronyltransferases, galacturonyltransferases,glucosaminyltransferases, N-glycolylneuraminyltransferases,rhamnosyltransferases, N-acetylrhamnosyltransferases,UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases,UDP-N-acetylglucosamine enolpyruvyl transferases andfucosaminyltransferases.
 9. The method according to claim 5, wherein thegalactosylated disaccharide or oligosaccharide is produced by a cell andthe cell is a metabolically engineered cell.
 10. The method according toclaim 5, wherein the galactosylated disaccharide or oligosaccharide isproduced by a cell and the cell is modified in the expression oractivity of an enzyme selected from the group consisting of glucosamine6-phosphate N-acetyltransferase, phosphatase, glycosyltransferase,L-glutamine D-fructose-6-phosphate aminotransferase, and UDP-glucose4-epimerase.
 11. The method according to claim 5, wherein thegalactosylated disaccharide or oligosaccharide is produced by a cell andthe cell is unable to convert N-acetylglucosamine-6-phosphate toglucosamine-6-phosphate, and/or unable to convertglucosamine-6-phosphate to fructose-6-phosphate.
 12. The methodaccording to claim 5, wherein the galactosylated disaccharide oroligosaccharide is produced by a cell and the cell is modified forenhanced UDP-galactose production and wherein the modification isselected from the group consisting of knock-out of a5′-nucleotidase/UDP-sugar hydrolase encoding gene or knock-out of agalactose-1-phosphate uridylyltransferase encoding gene.
 13. The methodaccording to claim 5, wherein the galactosylated disaccharide oroligosaccharide is produced by a cell and the cell uses at least oneprecursor for producing the galactosylated disaccharide oroligosaccharide, the precursor(s) being fed to the cell from thecultivation medium, and/or wherein the cell produces at least oneprecursor for producing galactosylated disaccharide or oligosaccharide.14. The method according to claim 13, wherein the precursor forproducing galactosylated disaccharide or oligosaccharide is completelyconverted into the galactosylated disaccharide or oligosaccharide. 15.The method according to claim 5, wherein the galactosylated disaccharideor oligosaccharide is produced by a cell and the cell produces thegalactosylated disaccharide or oligosaccharide intracellularly andwherein a fraction or substantially all of the produced galactosylateddisaccharide or oligosaccharide remains intracellularly and/or isexcreted outside the cell via passive or active transport.
 16. Themethod according to claim 5, wherein the galactosylated disaccharide oroligosaccharide is produced by a cell and the cell expresses a membranetransporter protein or a polypeptide having transport activity herebytransporting compounds across the outer membrane of the cell wall. 17.The method according to claim 16, wherein the membrane transporterprotein or polypeptide having transport activity: controls the flow overthe outer membrane of the cell wall of the galactosylated disaccharideor oligosaccharide and/or of at least one precursor and/or acceptor(s)to be used in the production of the galactosylated disaccharide oroligosaccharide, and/or provides improved production and/or enabledand/or enhanced efflux of the galactosylated disaccharide oroligosaccharide.
 18. The method according to claim 5, wherein thegalactosylated disaccharide or oligosaccharide is produced by a cell andthe cell comprises a modification for reduced production of acetatecompared to a non-modified progenitor, optionally the cell comprises alower or reduced expression and/or abolished, impaired, reduced ordelayed activity of any one or more of the proteins comprisingbeta-galactosidase, galactoside 0-acetyltransferase,N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphatedeaminase, N-acetylglucosamine repressor, ribonucleotidemonophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphateglucose-1-phosphate transferase, L-fuculokinase, L-fucose isomerase,N-acetylneuraminate lyase, N-acetylmannosamine kinase,N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man,EIID-Man, ushA, galactose-1-phosphate uridylyltransferase,glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase,ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobicrespiration control protein, transcriptional repressor IclR, lonprotease, glucose-specific translocating phosphotransferase enzyme IIBCcomponent ptsG, glucose-specific translocating phosphotransferase (PTS)enzyme IIBC component malX, enzyme IIAGlc, beta-glucoside specific PTSenzyme II, fructose-specific PTS multiphosphoryl transfer protein FruAand FruB, ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formatelyase, acetate kinase, phosphoacyltransferase, phosphateacetyltransferase, pyruvate decarboxylase compared to a non-modifiedprogenitor.
 19. The method according to claim 5, wherein thegalactosylated disaccharide or oligosaccharide is produced by a cell andthe cell is capable of producing phosphoenolpyruvate (PEP), optionallythe cell is modified for enhanced production and/or supply of PEPcompared to a non-modified progenitor.
 20. The method according to claim5, wherein the galactosylated disaccharide or oligosaccharide isproduced by a cell and the cell comprises a catabolic pathway forselected mono-, di- or oligosaccharides which is at least partiallyinactivated, the mono-, di-, or oligosaccharides being involved inand/or required for producing galactosylated disaccharide oroligosaccharide.
 21. The method according to claim 5, wherein thegalactosylated disaccharide or oligosaccharide is produced by a cell andthe cell resists a phenomenon of lactose killing when grown in anenvironment in which lactose is combined with one or more other carbonsource(s).
 22. The method according to claim 5, wherein thegalactosylated disaccharide or oligosaccharide is produced by a cell andthe cell produces 90 g/L or more of the galactosylated disaccharide oroligosaccharide in the whole broth and/or supernatant and/or wherein thegalactosylated disaccharide or oligosaccharide in the whole broth and/orsupernatant has a purity of at least 80% measured on the total amount ofthe galactosylated disaccharide or oligosaccharide and its precursor(s)in the whole broth and/or supernatant, respectively.
 23. The methodaccording to claim 5, wherein the galactosylated disaccharide oroligosaccharide is produced by a cell and wherein the conditionscomprise: use of a culture medium comprising at least one precursorand/or acceptor for producing galactosylated disaccharide oroligosaccharide, and/or adding to the culture medium at least oneprecursor and/or acceptor feed for producing galactosylated disaccharideor oligosaccharide.
 24. The method according to claim 5, wherein thegalactosylated disaccharide or oligosaccharide is produced by a cell,wherein the culture medium contains at least one precursor selected fromthe group consisting of lactose, galactose, fucose, and sialic acid. 25.The method according to claim 5, wherein the galactosylated disaccharideor oligosaccharide is produced by a cell, wherein a first phase ofexponential cell growth is provided by adding a carbon-based substrateto the culture medium comprising a precursor, followed by a second phasewherein: only a carbon-based substrate is added to the culture medium,or a carbon-based substrate and a precursor are added to the culturemedium.
 26. The method according to claim 5, wherein the galactosylateddisaccharide or oligosaccharide is produced by a cell and the cell iscapable of catabolizing a carbon source selected from the groupconsisting of glucose, fructose, mannose, galactose, lactose, sucrose,maltose, malto-oligosaccharides, trehalose, starch, cellulose,hemi-cellulose, corn-steep liquor, molasses, high-fructose syrup,glycerol, acetate, citrate, lactate, and pyruvate.
 27. The methodaccording to claim 5, wherein the galactosylated disaccharide oroligosaccharide is produced by a cell and the cell is a bacterium,fungus, yeast, a plant cell, an animal cell, or a protozoan cell. 28.The method according to claim 27, wherein the cell is a viableGram-negative bacterium that comprises a reduced or abolished synthesisof poly-N-acetyl-glucosamine (PNAG), enterobacterial common antigen(ECA), cellulose, colanic acid, core oligosaccharides, osmoregulatedperiplasmic glucans (OPG), glucosylglycerol, glycan, and/or trehalosecompared to a non-modified progenitor.
 29. The method according to claim2, wherein the method produces a mixture of charged and/or neutral di-and/or oligosaccharides comprising at least one galactosylateddisaccharide or oligosaccharide.
 30. The method according to claim 2,wherein the method produces a mixture of charged and/or neutraloligosaccharides comprising at least one galactosylated oligosaccharide.31. The method according to claim 5, wherein the galactosylateddisaccharide or oligosaccharide is produced by a cell and the cellproduces a mixture of charged and/or neutral di- and/or oligosaccharidescomprising at least one galactosylated disaccharide or oligosaccharide.32. The method according to claim 5, wherein the galactosylateddisaccharide or oligosaccharide is produced by a cell and the cellproduces a mixture of charged and/or neutral oligosaccharides comprisingat least one galactosylated oligosaccharide.
 33. The method according toclaim 3, wherein the separation comprises at least one of the followingsteps: clarification, ultrafiltration, nanofiltration, two-phasepartitioning, reverse osmosis, microfiltration, activated charcoal orcarbon treatment, treatment with non-ionic surfactants, enzymaticdigestion, tangential flow high-performance filtration, tangential flowultrafiltration, affinity chromatography, ion exchange chromatography,hydrophobic interaction chromatography and/or gel filtration, and ligandexchange chromatography.
 34. The method according to claim 3, furthercomprising purification of the galactosylated di- or oligosaccharide,optionally the purification comprises at least one of the followingsteps: use of activated charcoal or carbon, use of charcoal,nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment orion exchange, use of alcohols, use of aqueous alcohol mixtures,crystallization, evaporation, precipitation, drying, spray drying,lyophilization, spray freeze drying, freeze spray drying, band drying,belt drying, vacuum band drying, vacuum belt drying, drum drying, rollerdrying, vacuum drum drying, and vacuum roller drying.
 35. A cellmetabolically engineered to synthesize a galactosylated disaccharide oroligosaccharide by utilization of the N-acetylglucosamineb-1,X-galactosyltransferase of claim
 1. 36. The cell of claim 35,wherein the cell expresses any one of the N-acetylglucosamineb-1,3-galactosyltransferases and/or N-acetylglucosamineb-1,4-galactosyltransferases, is capable of synthesizing UDP-galactose(UDP-Gal) as donor for the galactosyltransferases, and is capable ofsynthesizing one or more acceptor(s) for the galactosyltransferases,wherein the acceptor(s) is/are an N-acetylglucosamine as amonosaccharide, and/or a di- or oligosaccharide having anN-acetylglucosamine and/or N-acetylgalactosamine at its non-reducingend.
 37. The cell of claim 35, wherein the cell is further capable ofsynthesizing one or more nucleotide-sugar donor(s) selected from thegroup consisting of GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal,UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine(UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc),UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose,UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc orUDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine,UDP-N-acetylfucosamine (UDP-L-FucNAc orUDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine(UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose),UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc orUDP-2-acetamido-26-dideoxy-L-glucose), GDP-L-quinovose,CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N₃,CMP-Neu4,5AC2, CMP-Neu5,7Ac₂, CMP-Neu5,9Ac₂, CMP-Neu5,7(8,9)Ac₂,UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, and UDP-xylose. 38.The cell of claim 35, wherein the cell is further capable of expressingat least one glycosyltransferase selected from the group consisting offucosyltransferases, sialyltransferases, galactosyltransferases,glucosyltransferases, mannosyltransferases,N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases,N-acetylmannosaminyltransferases, xylosyltransferases,glucuronyltransferases, galacturonyltransferases,glucosaminyltransferases, N-glycolylneuraminyltransferases,rhamnosyltransferases, N-acetylrhamnosyltransferases,UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases,UDP-N-acetylglucosamine enolpyruvyl transferases andfucosaminyltransferases.
 39. The cell of claim 35, wherein the cell ismodified in the expression or activity of an enzyme selected from thegroup consisting of glucosamine 6-phosphate N-acetyltransferase,phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphateaminotransferase, and UDP-glucose 4-epimerase.
 40. The cell of claim 35,wherein the cell is unable to convert N-acetylglucosamine-6-phosphate toglucosamine-6-phosphate, and/or unable to convertglucosamine-6-phosphate to fructose-6-phosphate.
 41. The cell of claim35, wherein the cell is modified for enhanced UDP-galactose productionand wherein the modification is selected from the group consisting ofknock-out of an 5′-nucleotidase/UDP-sugar hydrolase encoding gene, andknock-out of a galactose-1-phosphate uridylyltransferase encoding gene.42. The cell of claim 35, wherein the cell uses at least one precursorfor producing galactosylated disaccharide or oligosaccharide, theprecursor(s) being fed to the cell from the cultivation medium and/orwherein the cell is producing at least one precursor for producinggalactosylated disaccharide or oligosaccharide.
 43. The cell of claim42, wherein the precursor for producing galactosylated disaccharide oroligosaccharide is completely converted into the galactosylateddisaccharide or oligosaccharide.
 44. The cell of claim 35, wherein thecell produces the galactosylated disaccharide or oligosaccharideintracellularly and wherein a fraction or substantially all of theproduced galactosylated disaccharide or oligosaccharide remainsintracellularly and/or is excreted outside the cell via passive oractive transport.
 45. The cell of claim 35, wherein the cell expresses amembrane transporter protein or a polypeptide having transport activityhereby transporting compounds across the outer membrane of the cellwall.
 46. The cell of claim 45, wherein the membrane transporter proteinor polypeptide having transport activity: controls the flow over theouter membrane of the cell wall of the galactosylated disaccharide oroligosaccharide and/or of at least one precursor and/or acceptor(s) tobe used in the production of the galactosylated disaccharide oroligosaccharide, and/or provides improved production and/or enabledand/or enhanced efflux of the galactosylated disaccharide oroligosaccharide.
 47. The cell of claim 35, wherein the cell comprises amodification for reduced production of acetate compared to anon-modified progenitor, optionally the cell comprises a lower orreduced expression and/or abolished, impaired, reduced or delayedactivity of any one or more of the proteins comprisingbeta-galactosidase, galactoside 0-acetyltransferase,N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphatedeaminase, N-acetylglucosamine repressor, ribonucleotidemonophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphateglucose-1-phosphate transferase, L-fuculokinase, L-fucose isomerase,N-acetylneuraminate lyase, N-acetylmannosamine kinase,N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man,EIID-Man, ushA, galactose-1-phosphate uridylyltransferase,glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase,ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobicrespiration control protein, transcriptional repressor IclR, lonprotease, glucose-specific translocating phosphotransferase enzyme IIBCcomponent ptsG, glucose-specific translocating phosphotransferase (PTS)enzyme IIBC component malX, enzyme IIAGlc, beta-glucoside specific PTSenzyme II, fructose-specific PTS multiphosphoryl transfer protein FruAand FruB, ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formatelyase, acetate kinase, phosphoacyltransferase, phosphateacetyltransferase, and pyruvate decarboxylase compared to a non-modifiedprogenitor.
 48. The cell of claim 35, wherein the cell is capable ofproducing phosphoenolpyruvate (PEP), optionally the cell is modified forenhanced production and/or supply of PEP compared to a non-modifiedprogenitor.
 49. The cell of claim 35, wherein the cell comprises acatabolic pathway for selected mono-, di- or oligosaccharides which isat least partially inactivated, the mono-, di-, or oligosaccharidesbeing involved in and/or required for producing galactosylateddisaccharide or oligosaccharide.
 50. The cell of claim 35, wherein thecell resists the phenomenon of lactose killing when grown in anenvironment in which lactose is combined with one or more other carbonsource(s).
 51. The cell of claim 35, wherein the cell is capable ofcatabolizing a carbon source selected from the group consisting ofglucose, fructose, mannose, galactose, lactose, sucrose, maltose,malto-oligosaccharides, trehalose, starch, cellulose, hemi-cellulose,molasses, corn-steep liquor, high-fructose syrup, glycerol, acetate,citrate, lactate, and pyruvate.
 52. The cell of claim 35, wherein thecell is a bacterium, fungus, yeast, a plant cell, an animal cell, or aprotozoan cell.
 53. The cell of claim 52, wherein the cell is a viableGram-negative bacterium that comprises a reduced or abolished synthesisof poly-N-acetyl-glucosamine (PNAG), enterobacterial common antigen(ECA), cellulose, colanic acid, core oligosaccharides, osmoregulatedperiplasmic glucans (OPG), glucosylglycerol, glycan, and/or trehalosecompared to a non-modified progenitor.
 54. The cell of claim 35, whereinthe cell produces a mixture of charged and/or neutral di- and/oroligosaccharides comprising at least one galactosylated disaccharide oroligosaccharide.
 55. The cell of claim 35, wherein the cell produces amixture of charged and/or neutral oligosaccharides comprising at leastone galactosylated oligosaccharide.
 56. A method of producing agalactosylated disaccharide or oligosaccharide, the method comprising:cultivating the cell of claim 35 so as to produce a galactosylateddisaccharide or oligosaccharide.
 57. A method of producing a mixture ofcharged and/or neutral di- and/or oligosaccharides comprising at leastone galactosylated disaccharide or oligosaccharide, the methodcomprising: cultivating the cell of claim 35 so as to produce a mixtureof charged and/or neutral di- and/or oligosaccharides comprising atleast one galactosylated disaccharide or oligosaccharide.
 58. A methodof producing a mixture of charged and/or neutral oligosaccharidescomprising at least one galactosylated oligosaccharide, the methodcomprising: cultivating the cell of claim 35 so as to produce a mixtureof charged and/or neutral oligosaccharides comprising at least onegalactosylated oligosaccharide.